Gene therapy for effector cell regulation

ABSTRACT

The present invention provides a nucleic acid-based therapeutic composition to treat an animal with disease by controlling the activity of effector cells, including T cells, macrophages, monocytes and/or natural killer cells, in the animal. Therapeutic compositions of the present invention include superantigen-encoding nucleic acid molecules, either in the presence or absence of a cytokine-encoding nucleic acid molecule and/or chemokine-encoding nucleic acid molecules, depending upon the disease being treated. The present invention also relates to an adjuvant for use with nucleic acid-based vaccines. Adjuvant compositions of the present invention include an immunogen combined with superantigen-encoding nucleic acid molecules, either in the presence or absence of a cytokine-encoding nucleic acid molecule and/or chemokine-encoding nucleic acid molecules.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a continuation-in-part of U.S. patentapplication Ser. No. 08/446,918 for "Gene Therapy for T CellRegulation", filed May 18, 1995, U.S. Pat. No. 5,705,151 incorporatedherein by this reference in its entirety. The present application isalso a continuation-in-part of U.S. patent application Ser. No.08/484,169 for "Mycobacterium Peptides, Nucleic Acid Molecules, and UsesThereof", filed Jun. 7, 1995, now abandoned, incorporated herein by thisreference in its entirety.

FIELD OF THE INVENTION

The present invention relates to a product and process for regulating Tcell activity by providing a superantigen gene, in the presence orabsence of a cytokine and/or chemokine gene. The present invention alsorelates to a product and process for regulating T cell activity byproviding a peptide and a superantigen gene, in the presence or absenceof a cytokine and/or chemokine gene. In particular, the presentinvention relates to a product and process for controlling tumordevelopment, immune responses to infectious diseases and diseases causedby immunological disorders.

BACKGROUND OF THE INVENTION

Two major causes of disease include infectious agents and malfunctionsof normal biological functions of an animal. Examples of infectiousagents include viruses, bacteria, parasites, yeast and other fungi.Examples of abnormal biological function include uncontrolled cellgrowth, abnormal immune responses and abnormal inflammatory responses.Traditional reagents used attempt to protect an animal from diseaseinclude reagents that destroy infectious agents or cells involved inderegulated biological functions. Such reagents, however, can result inunwanted side effects. For example, anti-viral drugs that disrupt thereplication of viral DNA also often disrupt DNA replication in normalcells in the treated patient. Other treatments with chemotherapeuticreagents to destroy cancer cells typically leads to side effects, suchas bleeding, vomiting, diarrhea, ulcers, hair loss and increasedsusceptibility to secondary cancers and infections.

An alternative method of disease treatment includes modulating theimmune system of a patient to assist the patient's natural defensemechanisms. Traditional reagents and methods used to attempt to regulatean immune response in a patient also result in unwanted side effects andhave limited effectiveness. For example, immunosuppressive reagents(e.g., cyclosporin A, azathioprine, and prednisone) used to treatpatients with autoimmune disease also suppress the patient's entireimmune response, thereby increasing the risk of infection. In addition,immunopharmacological reagents used to treat cancer (e.g., interleukins)are short-lived in the circulation of a patient and are ineffectiveexcept in large doses. Due to the medical importance of immuneregulation and the inadequacies of existing immunopharmacologicalreagents, reagents and methods to regulate specific parts of the immunesystem have been the subject of study for many years.

Stimulation or suppression of the immune response in a patient can be aneffective treatment for a wide variety of medical disorders. Tlymphocytes (T cells) are one of a variety of distinct cell typesinvolved in an immune response. The activity of T cells is regulated byantigen, presented to a T cell in the context of a majorhistocompatibility complex (MHC) molecule. The T cell receptor (TCR)then binds to the MHC:antigen complex. Once antigen is complexed to MHC,the MHC:antigen complex is bound by a specific TCR on a T cell, therebyaltering the activity of that T cell.

The use of certain staphylococcal enterotoxin proteins that are capableof complexing with MHC molecules to influence T cell function has beensuggested by various investigators, including, for example, White etal., Cell 56:27-35, 1989; Rellahan et al. J. Expt. Med. 172:1091-1100,1990; Micusan et al., Immunology 5:3-11, 1993; Hermann et al.,Immunology 5:33-39, 1993; Bhardwaj et al., J. Expt. Med. 178:633-642,1993; and Kalland et al., Med. Oncol. & Tumor Pharmacother., 10:37-47,1993. In particular, various investigators have suggested thatStaphylococcal enterotoxin proteins are useful for treating tumors,including Newell et al., Proc. Natl. Acad. Sci. USA 88:1074-1078, 1991;Kalland et al., PCT Application No. WO 91/04053, published Apr. 4, 1991;Dohlstein et al., Proc. Natl. Acad. Sci. USA 88:9287-9291, 1991; Hedlundet al., Cancer Immunol. Immunother. 36:89-93, 1993; Lando et al., CancerImmunol. Immunother. 36:223-228, 1993; Lukacs et al., J. Exp. Med.178:343-348, 1993; Ochi et al., J. Immunol. 151:3180-3186, 1993; andTerman et al., PCT Application No. WO 93/24136, published Dec. 9, 1993.These investigators, however, have only disclosed the use of bacterialenterotoxin proteins themselves. The use of bacterial enterotoxinprotein has the major disadvantage of being toxic to the recipient ofthe protein.

Thus, there is a need for a product and process that allows for thetreatment of disease using bacterial enterotoxins in a non-toxic manner.

SUMMARY

Traditional pharmaceutical reagents used to treat cancer, infectiousdiseases and diseases caused by immunological disorders often haveharmful side effects. In addition, such reagents can be unpredictable(e.g., treatment of cancer, vaccination against infectious agents). Forexample, chemotherapy and radiotherapy often cause extensive normaltissue damage during the process of treating cancerous tissue. Inaddition, vaccine treatments for the prevention or cure of infectiousdiseases are often ineffective because adjuvants useful in vaccinetherapy are toxic to an animal.

The present invention is particularly advantageous in that it providesan effective therapeutic composition that enables the safe treatment ofan animal with a reagent that is a potentially toxic an immunogenicprotein. Upon delivery, expression of acid molecules contained in thetherapeutic composition result in localized production of an effectivebut non-toxic amount of encoded proteins that may be toxic atconcentrations that would be required if the encoded proteins wereadministered directly. The therapeutic compositions of the presentinvention can provide long term expression of the encoded proteins at asite in an animal. Such long term expression allows for the maintenanceof an effective, but non-toxic, dose of the encoded protein to treat adisease and limits the frequency of administration of the therapeuticcomposition needed to treat an animal. In addition, because of the lackof toxicity, therapeutic compositions of the present invention can beused in repeated treatments.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 illustrates the expression of superantigen-encoding DNA plasmidsin mammalian cells.

FIG. 2 illustrates the proliferative response of canine PBMC's to caninemelanoma cells transfected with a superantigen-encoding DNA plasmids.

FIGS. 3A and 3B illustrate the release of superantigen protein by CHOcells transfected with superantigen-encoding DNA plasmids.

FIG. 4 illustrates the proliferative response of the Vβ3+ T cell cloneAD10 to melanoma cells transfected with superantigen-encoding DNAplasmid.

FIG. 5 illustrates the release of canine GM-CSF by CHO cells transfectedwith GM-CSF-encoding DNA plasmid.

FIGS. 6A and 6B illustrate the vaccination of mice with autologous tumorcells transfected with superantigen-encoding DNA plasmid.

FIG. 7 illustrates the effect of tumor target transfection on cytotoxicT cell lysis.

FIG. 8 illustrates the response of Vβ3+ T cells to intramuscularinjection of a superantigen-encoding DNA plasmid.

FIG. 9 illustrates the antibody response resulting from theco-administration of DNA encoding an adjuvant and DNA encodingovalbumin.

FIG. 10 illustrates that the co-administration of DNA encoding anadjuvant and DNA encoding ovalbumin increase interferon-gamma releasefrom T cells restimulated in vitro by the ovalbumin protein.

FIG. 11 illustrates that the co-administration of DNA encoding anadjuvant and DNA encoding ovalbumin increase T cell proliferativeresponses to ovalbumin.

FIG. 12 illustrates that the co-administration of DNA encoding anadjuvant and DNA encoding ovalbumin increases CTL responses toovalbumin.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to a novel product and process forcontrolling effector cell activity. The present invention also relatesto a novel adjuvant useful for enhancing an immune response. It is nowknown for the first time that a composition containing nucleic acidmolecules encoding a superantigen, rather than superantigen proteins, isan effective therapeutic reagent for treating disease and is aneffective adjuvant for enhancing an immune response. As used herein, adisease refers to any biological abnormality that is not beneficial to asubject. The present inventors have also discovered that administrationof a combination of nucleic acid molecules encoding: (1) a superantigen;(2) a superantigen and a cytokine; or (3) a superantigen and achemokine, can act synergistically to effectively treat cancer andinfectious disease. The present invention includes therapeuticcompositions comprising: (a) an isolated nucleic acid molecule encodinga superantigen; or (b) an isolated nucleic acid molecule encoding asuperantigen in combination with an isolated nucleic acid moleculeencoding a cytokine and/or an isolated nucleic acid molecule encoding achemokine. Administration of a therapeutic composition of the presentinvention to an animal results in the production of superantigen,cytokine or chemokine proteins, referred to herein as "encodedproteins." Each of the components of a therapeutic composition of thepresent invention is described in detail below, followed by adescription of the methods by which the therapeutic composition is usedand delivered.

One embodiment of the present invention includes a method for increasingeffector cell immunity in an animal, the method comprising administeringto an animal an effective amount of a therapeutic compositioncomprising: (a) an isolated nucleic acid molecule encoding asuperantigen; or (b) an isolated nucleic acid molecule encoding asuperantigen in combination with an isolated nucleic acid moleculeencoding a cytokine and/or an isolated nucleic acid molecule encodingchemokine. According to the present embodiment, the nucleic acidmolecules are operatively linked to one or more transcription controlsequences and the therapeutic composition is targeted to a site in theanimal that contains an abnormal cell. According to the presentinvention, an effector cell, includes a helper T cell, a cytotoxic Tcell, a macrophage, a monocyte and/or a natural killer cell. Forexample, the method of the present invention can be performed toincrease the number of effector cells in an animal that are capable ofkilling or releasing cytokines or chemokines when presented withantigens derived from an abnormal cell or a pathogen. An effectiveamount of a therapeutic composition of the present invention comprisesan amount capable of treating a disease as described herein.Alternatively, a method of the present invention can be performed todecrease the number of T cells found in a T cell subset that ispreferentially stimulated and expanded by an autoantigen.

As used herein, effector cell immunity refers to increasing the numberand/or the activity of effector cells in the area of the abnormal cell.In particular, T cell activity refers to increasing the number and/orthe activity of T cells in the area of the abnormal cell. Also, as usedherein, an abnormal cell refers to a cell displaying abnormal biologicalfunction, such as abnormal growth, development or death. Abnormal cellsof the present invention, preferably includes cancer cells, cellsinfected with an infectious agent (i.e., a pathogen) and non-cancerouscells having abnormal proliferative growth (e.g., sarcoidosis,granulomatous disease or papillomas) and with cancer cells and infectedcells.

Another embodiment of the present invention is a method to treat ananimal with cancer, the method comprising administering to an animal aneffective amount of a therapeutic composition comprising: (a) a nucleicacid molecule encoding a superantigen; or (b) a nucleic acid moleculeencoding a superantigen in combination with an isolated nucleic acidmolecule encoding a cytokine and/or a nucleic acid molecule encoding achemokine. According to the present embodiment, the nucleic acidmolecules are operatively linked to one or more transcription controlsequences and the therapeutic composition is targeted to the site of acancer.

One embodiment of a therapeutic composition of the present inventioncomprises an isolated nucleic acid molecule encoding a superantigen(also referred to herein as a "superantigen-encoding" nucleic acidmolecule). Another embodiment of a therapeutic composition of thepresent invention comprises an isolated nucleic acid molecule encoding asuperantigen, combined with an isolated nucleic acid molecule encoding acytokine (also referred to herein as a "cytokine-encoding" nucleic acidmolecule) and/or a nucleic acid molecule encoding a chemokine (alsoreferred to as a "chemokine-encoding" nucleic acid molecule). Accordingto these embodiments, the nucleic acid molecules are operatively linkedto one or more transcription control sequences. It is to be noted thatthe term "a" or "an" entity refers to one or more of that entity; forexample, a compound refers to one or more compounds. As such, the terms"a" (or "an"), "one or more" and "at least one" can be usedinterchangeably herein. According to the present invention, an isolated,or biologically pure, nucleic acid molecule, is a nucleic acid moleculethat has been removed from its natural milieu. As such, "isolated" and"biologically pure" do not necessarily reflect the extent to which thenucleic acid molecule has been purified. An isolated nucleic acidmolecule can include DNA, RNA, or derivatives of either DNA or RNA. Anisolated superantigen or cytokine nucleic acid molecule can be obtainedfrom its natural source, either as an entire (i.e., complete) gene or aportion thereof capable of encoding a superantigen protein capable ofbinding to an MHC molecule or a cytokine protein capable of binding to acomplementary cytokine receptor. A nucleic acid molecule can also beproduced using recombinant DNA technology (e.g., polymerase chainreaction (PCR) amplification, cloning) or chemical synthesis. Nucleicacid molecules include natural nucleic acid molecules and homologuesthereof, including, but not limited to, natural allelic variants andmodified nucleic acid molecules in which nucleotides have been inserted,deleted, substituted, and/or inverted in such a manner that suchmodifications do not substantially interfere with the nucleic acidmolecule's ability to encode a functional superantigen or a functionalcytokine of the present invention.

A nucleic acid molecule homologue can be produced using a number ofmethods known to those skilled in the art (see, for example, Sambrook etal., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor LabsPress, 1989). For example, nucleic acid molecules can be modified usinga variety of techniques including, but not limited to, classicmutagenesis techniques and recombinant DNA techniques, such assite-directed mutagenesis, chemical treatment of a nucleic acid moleculeto induce mutations, restriction enzyme cleavage of a nucleic acidfragment, ligation of nucleic acid fragments, polymerase chain reaction(PCR) amplification and/or mutagenesis of selected regions of a nucleicacid sequence, synthesis of oligonucleotide mixtures and ligation ofmixture groups to "build" a mixture of nucleic acid molecules andcombinations thereof. Nucleic acid molecule homologues can be selectedfrom a mixture of modified nucleic acids by screening for the functionof the protein encoded by the nucleic acid (e.g., superantigen, cytokineor chemokine activity, as appropriate). Techniques to screen forsuperantigen, cytokine or chemokine activity are known to those of skillin the art.

Although the phrase "nucleic acid molecule" primarily refers to thephysical nucleic acid molecule and the phrase "nucleic acid sequence"primarily refers to the sequence of nucleotides on the nucleic acidmolecule, the two phrases can be used interchangeably, especially withrespect to a nucleic acid molecule, or a nucleic acid sequence, beingcapable of encoding a superantigen, a cytokine or a chemokine protein.In addition, the phrase "recombinant molecule" primarily refers to anucleic acid molecule operatively linked to a transcription controlsequence, but can be used interchangeably with the phrase "nucleic acidmolecule" which is administered to an animal. As heretofore disclosed,superantigen or cytokine proteins of the present invention include, butare not limited to, proteins having full-length superantigen, cytokineor chemokine coding regions, proteins having partial superantigenregions capable of binding to an MHC molecule, cytokine coding regionscapable of binding to a complementary cytokine receptor, chemokinecoding regions capable of binding to a complementary chemokine receptor,fusion proteins and chimeric proteins comprising combinations ofdifferent superantigens, cytokines and/or chemokines.

One embodiment of the present invention is an isolatedsuperantigen-encoding nucleic acid molecule that encodes at least aportion of a full-length superantigen, or a homologue of a superantigen.As used herein, "at least a portion of a superantigen" refers to aportion of a superantigen protein capable of binding to an MHC moleculein such a manner that a TCR can bind to the resulting superantigen:MHCcomplex. Preferably, a superantigen nucleic acid molecule of the presentinvention encodes an entire coding region of a superantigen, and morepreferably the coding region absent a leader sequence. Production of atruncated superantigen protein lacking a bacterial leader sequence ispreferred to enhance secretion of the superantigen from a cell. As usedherein, a homologue of a superantigen is a protein having an amino acidsequence that is sufficiently similar to a natural superantigen aminoacid sequence that a nucleic acid sequence encoding the homologueencodes a protein capable of binding to an MHC molecule.

In accordance with the present invention, a superantigen comprises afamily of T cell regulatory proteins that are capable of binding both toan MHC molecule. A superantigen binds to the extracellular portion of anMHC molecule to form and MHC:superantigen complex. The activity of a Tcell can be modified when a TCR binds to an MHC:superantigen complex.Under certain circumstances, an MHC:superantigen complex can have amitogenic role (i.e., the ability to stimulate the proliferation of Tcells) or a suppressive role (i.e., deletion of T cell subsets). Theability of an MHC:superantigen complex to have a stimulatory orsuppressive role can depend upon factors, such as the concentration andenvironment (i.e., tissue location and/or the presence of cytokines).

The mitogenic role of a superantigen is distinct from other knownmitogens (e.g., lectins derived from plants) in that superantigens arecapable of stimulating the proliferation of particular subsets of Tcells having TCR's that specifically bind to the superantigen. Forexample, a superantigen, when added to a mixed lymphocyte population, isable to stimulate the proliferation of a select population of T cellsfrom the mixed population of cells. Examples of T cell subsetsstimulated by superantigens complexed with MHC molecules include T cellsexpressing a TCR comprising mouse V.sub.β 1, V.sub.β 3, V.sub.β 7,V.sub.β 8.1, V.sub.β 8.2, V.sub.β 8.3, V.sub.β 10, V.sub.β 11, V.sub.β17, V.sub.β 15 or V.sub.β 16 chains, and T cells expressing a TCRcomprising human V.sub.β 1.1, V.sub.β 2, V.sub.β 3, V.sub.β 5, V.sub.β6, V.sub.β 7.3, V.sub.β 7.4, V.sub.β 9.1, V.sub.β 12, V.sub.β 14,V.sub.β 15, V.sub.β 17 or V.sub.β 20 chains.

A superantigen-encoding nucleic acid molecule of the present inventionpreferably encodes superantigens that includes, but is not limited to,staphylococcal enterotoxins, retroviral antigens, streptococcalantigens, mycoplasma antigens, mycobacterium antigens, viral antigens(e.g., a superantigen from mouse mammary tumor virus, rabies virus orherpes virus) and endoparasitic antigens (e.g., protozoan or helminthantigens), more preferably staphylococcal enterotoxins, and even morepreferably Staphylococcal enterotoxin A (SEA), Staphylococcalenterotoxin B (SEB), Staphylococcal enterotoxin C₁ (SEC₁),Staphylococcal enterotoxin C₂ (SEC₂), Staphylococcal enterotoxin C₃(SEC₃), Staphylococcal enterotoxin D (SED), Staphylococcal enterotoxin E(SEE) and Toxic Shock Syndrome Toxin (TSST).

A preferred nucleic acid molecule encoding a Staphylococcal enterotoxinof the present invention comprises a nucleic acid sequence representedby SEQ ID NO:1 (representing a full-length SEB gene), SEQ ID NO:3(representing a full-length SEA gene) or SEQ ID NO:6 (representing afull-length TSST gene). A preferred Staphylococcal enterotoxin proteinof the present invention comprises an amino acid sequence represented bySEQ ID NO:2 (representing a full-length SEB protein), SEQ ID NO:4(representing a full-length SEA protein) or SEQ ID NO:7 (representing afull-length TSST protein).

In a preferred embodiment, a nucleic acid molecule of the presentinvention encoding a superantigen comprises a nucleic acid sequencespanning base pair 46 to at least base pair 768 of SEQ ID NO:1, anucleic acid sequence spanning base pair 46 to about base pair 751 ofSEQ ID NO:3 or SEQ ID NO:6.

Another embodiment of the present invention includes a cytokine-encodingnucleic acid molecule that encodes a full-length cytokine or a homologueof the cytokine protein. As used herein, a homologue of a cytokine is aprotein having an amino acid sequence that is sufficiently similar to anatural cytokine amino acid sequence so as to have cytokine activity. Inaccordance with the present invention, a cytokine includes a proteinthat is capable of affecting the biological function of another cell. Abiological function affected by a cytokine can include, but is notlimited to, cell growth, cell differentiation or cell death. Preferably,a cytokine of the present invention is capable of binding to a specificreceptor on the surface of a cell, thereby affecting the biologicalfunction of a cell.

A cytokine-encoding nucleic acid molecule of the present inventionencodes a cytokine that is capable of affecting the biological functionof a cell, including, but not limited to, a lymphocyte, a muscle cell, ahematopoietic precursor cell, a mast cell, a natural killer cell, amacrophage, a monocyte, an epithelial cell, an endothelial cell, adendritic cell, a mesenchymal cell, a Langerhans cell, cells found ingranulomas and tumor cells of any cellular origin, and more preferably amesenchymal cell, an epithelial cell, an endothelial cell, a musclecell, a macrophage, a monocyte, a T cell and a dendritic cell.

A preferred cytokine nucleic acid molecule of the present inventionencodes a hematopoietic growth factor, an interleukin, an interferon, animmunoglobulin superfamily molecule, a tumor necrosis factor familymolecule and/or a chemokine (i.e., a protein that regulates themigration and activation of cells, particularly phagocytic cells). Amore preferred cytokine nucleic acid molecule of the present inventionencodes a granulocyte macrophage colony stimulating factor (GM-CSF),tumor necrosis factor α (TNF-α), macrophage colony stimulating factor(M-CSF), interleukin-1 (IL-1), interleukin-6 (IL-6), interleukin-12(IL-12) and/or IGIF. An even more preferred cytokine nucleic acidmolecule of the present invention encodes GM-CSF, IL-12, IGIF and/orTNF-α, with GM-CSF being even more preferred.

As will be apparent to one of skill in the art, the present invention isintended to apply to cytokines derived from all types of animals. Apreferred animal from which to derive cytokines includes a mouse, ahuman, a cat and a dog. A more preferred animal from which to derivecytokines includes a cat, a dog and a human. An even more preferredanimal from which to derive cytokines is a human.

According to the present invention, a cytokine-encoding nucleic acidmolecule of the present invention is derived from the same species ofanimal as the animal to be treated. For example, a cytokine-encodingnucleic acid molecule derived from a canine (i.e., dog) nucleic acidmolecule is used to treat a disease in a canine. Thus, a preferredcytokine-encoding nucleic acid molecule of the present inventioncomprises a nucleic acid molecule encoding human GM-CSF, as described inthe art. A human GM-CSF-encoding nucleic acid molecule of the presentinvention can be produced using methods standard PCR amplificationmethods with primers designed from the human GM-CSF nucleic acidsequence disclosed in Nash (Blood 78:930, 1991). Such PCR products canbe cloned into a PCR₃ expression vector using the methods generallydescribed in Example 1.

Another embodiment of the present invention includes achemokine-encoding nucleic acid molecule that encodes a full-lengthchemokine or a homologue of the chemokine protein. As used herein, ahomologue of a chemokine is a protein having an amino acid sequence thatis sufficiently similar to a natural chemokine amino acid sequence so asto have chemokine activity. In accordance with the present invention, achemokine includes a protein that is capable of attracting cellsinvolved in an immune response (immunologic cells), including phagocyticcells. For example, immunologic cells are recruited from the blood to asite at which the chemokine is located (e.g., a site of infection).Preferably, a chemokine of the present invention is capable of bindingto a specific receptor on the surface of a cell, thereby attracting thecell to a specific location.

A chemokine-encoding nucleic acid molecule of the present inventionencodes a chemokine that is capable of attracting a cell to a site,including, but not limited to, a dendritic cell, a neutrophil, amacrophage, a T lymphocyte and Langerhans cells, and more preferably adendritic cell, a macrophage and a T lymphocyte.

A preferred chemokine-encoding nucleic acid molecule of the presentinvention encodes an α-chemokine or a β-chemokine. A more preferredchemokine-encoding nucleic acid molecule of the present inventionencodes a C5a, interleukin-8 (IL-8) , monocyte chemotactic protein 1α(MIP1α), monocyte chemotactic protein 1β (MIP1β), monocytechemoattractant protein 1 (MCP-1), monocyte chemoattractant protein 3(MCP-3), platelet activating factor (PAFR), N-Formyl-methionyl-leucyl^(3H) !phenylalanine (FMLPR), leukotriene B₄ (LTB₄ R), gastrin releasingpeptide (GRP), RANTES, eotaxin, lymphotactin, IP10, I-309, ENA78, GCP-2,NAP-2 and/or MGSA/gro. An even more preferred chemokine-encoding nucleicacid molecule of the present invention encodes IL-8, MIP1α, MIP1β,MCP-1, MCP-3, RANTES and/or NAP-2, with IL-8, Rantes, MIP1α and/or MIP1βbeing even more preferred.

As will be apparent to one of skill in the art, the present invention isintended to apply to chemokines derived from all types of animals.Preferred animals from which to derive chemokines includes mice, humans,dogs, cats, cattle and horses. More preferred animals from which toderive chemokines includes dogs, cats, humans and cattle. Even morepreferred animals from which to derive chemokines are humans.

According to the present invention, a chemokine-encoding nucleic acidmolecule of the present invention is derived from the same species ofanimal as the animal to be treated. For example, a chemokine-encodingnucleic acid molecule derived from a canine (i.e., dog) nucleic acidmolecule is used to treat a disease in a canine. Thus, a preferredchemokine-encoding nucleic acid molecule of the present inventioncomprises a nucleic acid molecule encoding a dog, cat, human, bovineand/or equine chemokine. Preferred nucleic acid molecules of the presentinvention encode IL-8, Rantes, MIP1α and/or MIP1β, as described in theart. For example, a human MIP1α-encoding nucleic acid molecule of thepresent invention can be produced using standard PCR amplificationmethods with primers designed from the human MIP1α-encoding nucleic acidsequence disclosed in the art. Such PCR products can be cloned into aPCR₃ expression vector using the methods generally described in Example1.

The present invention includes a nucleic acid molecule of the presentinvention operatively linked to one or more transcription controlsequences to form a recombinant molecule. The phrase "operativelylinked" refers to linking a nucleic acid molecule to a transcriptioncontrol sequence in a manner such that the molecule is able to beexpressed when transfected (i.e., transformed, transduced ortransfected) into a host cell. Transcription control sequences aresequences which control the initiation, elongation, and termination oftranscription. Particularly important transcription control sequencesare those which control transcription initiation, such as promoter,enhancer, operator and repressor sequences. Suitable transcriptioncontrol sequences include any transcription control sequence that canfunction in at least one of the recombinant cells of the presentinvention. A variety of such transcription control sequences are knownto those skilled in the art. Preferred transcription control sequencesinclude those which function in animal, bacteria, helminth, insectcells, and preferably in animal cells. More preferred transcriptioncontrol sequences include, but are not limited to, simian virus 40(SV-40), β-actin, retroviral long terminal repeat (LTR), Rous sarcomavirus (RSV), cytomegalovirus (CMV), tac, lac, trp, trc, oxy-pro,omp/lpp, rrnB, bacteriophage lambda (λ) (such as λp_(L) and λp_(R) andfusions that include such promoters), bacteriophage T7, T7lac,bacteriophage T3, bacteriophage SP6, bacteriophage SP01,metallothionein, alpha mating factor, Pichia alcohol ooxidase,alphavirus subgenomic promoters (such as Sindbis virus subgenomicpromoters), baculovirus, Heliothis zea insect virus, vaccinia virus andother poxviruses, herpesvirus, and adenovirus transcription controlsequences, as well as other sequences capable of controlling geneexpression in eukaryotic cells. Additional suitable transcriptioncontrol sequences include tissue-specific promoters and enhancers (e.g.,tumor cell-specific enhancers and promoters), and inducible promoters(e.g., tetracycline). Transcription control sequences of the presentinvention can also include naturally occurring transcription controlsequences naturally associated with a gene encoding a superantigen, acytokine or a chemokine of the present invention.

Recombinant molecules of the present invention, which can be either DNAor RNA, can also contain additional regulatory sequences, such astranslation regulatory sequences, origins of replication, and otherregulatory sequences that are compatible with the recombinant cell. Inone embodiment, a recombinant molecule of the present invention alsocontains secretory signals (i.e., signal segment nucleic acid sequences)to enable an expressed superantigen, cytokine or a chemokine protein tobe secreted from the cell that produces the protein. Suitable signalsegments include: (1) a bacterial signal segment, in particular asuperantigen signal segment; (2) a cytokine signal segment; (3) achemokine signal segment; (4) or any heterologous signal segment capableof directing the secretion of a superantigen, cytokine and/or chemokineprotein of the present invention. Preferred signal segments include, butare not limited to, signal segments associated with SEB, SEA, TSST,GM-CSF, M-CSF, TNFα, IL-1, IL-6, IL-12 C5a, IGIF, IL-8, MIP1α, MIP1β,MCP-1, MCP-3, PAFR, FMLPR, LTB₄ R, GRP, RANTES, eotaxin, lymphotactin,IP10, I-309, ENA78, GCP-2, NAP-2 and/or MGSA/gro protein.

Preferred recombinant molecules of the present invention include arecombinant molecule containing a nucleic acid molecule encoding asuperantigen, a recombinant molecule containing a nucleic acid moleculeencoding a cytokine, a recombinant molecule containing a nucleic acidmolecule encoding a chemokine, a recombinant molecule containing anucleic acid molecule encoding a superantigen and a nucleic acidmolecule encoding a cytokine to form a chimeric recombinant molecule, ora recombinant molecule containing a nucleic acid molecule encoding asuperantigen and a nucleic acid molecule encoding a chemokine to form achimeric recombinant molecule. The nucleic acid molecules contained insuch recombinant chimeric molecules are operatively linked to one ormore transcription control sequences, in which each nucleic acidmolecule contained in a chimeric recombinant molecule can be expressedusing the same or different regulatory control sequences. Preferredrecombinant molecules of the present invention comprise a nucleic acidsequence represented by SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO:5, orcombinations thereof. Particularly preferred recombinant moleculesinclude PCR₃ -SEB, PCR₃ -SEA, PCR₃ -SEB.S, PCR₃ -SEA.S, PCR₃ -TSST andPCR₃ -GM₃, the production of which is disclosed herein. Other preferrednucleic acid sequences include Rantes nucleic acid sequence (SEQ ID.NO:13), MIP1α nucleic acid sequence (see Davatelis et al., J. Exp. Med.167:1939-1944, 1988) and MIP1β, nucleic acid sequence (see Sherry etal., J. Exp. Med. 168:2251-2259, 1988).

According to the present invention, a recombinant molecule can bedicistronic. A cistron refers to a unit of DNA that is capable ofencoding an amino acid sequence having a naturally-occurring biologicalfunction. A dicistronic plasmid refers to a plasmid containing 2cistrons. Preferably, a dicistronic recombinant molecule of the presentinvention comprises an internal ribosome entry site (IRES) element towhich eukaryotic ribosomes can bind (see, for example, Jang et al., J.Virol. 62:2636-2643, 1988; Pelletier et al. Nature 334:320-325, 1988;Jackson, Nature 353:14-15, 1991; Macejek et al., Nature 353:90-94, 1991;Oh et al., Genes & Develop. 6:1643-1653, 1992; Molla et al., Nature356:255-257, 1992; and Kozak, Crit. Rev. Biochem. Molec. Biol.27(4,5):385-402, 1992).

In one embodiment, a dicistronic recombinant molecule of the presentinvention comprises a eukaryotic promoter, operatively linked to asuperantigen-encoding nucleic acid molecule of the present invention anda cytokine-encoding nucleic acid molecule of the present inventionseparated by an IRES nucleic acid sequence, or a superantigen-encodingnucleic acid molecule of the present invention and chemokine-encodingnucleic acid molecule of the present invention separated by an IRESnucleic acid sequence.

In another embodiment, a dicistronic recombinant molecule of the presentinvention comprises a eukaryotic promoter, operatively linked to a firstsuperantigen-encoding nucleic acid molecule of the present invention anda second superantigen-encoding nucleic acid molecule of the presentinvention separated by an IRES nucleic acid sequence. Preferably, afirst superantigen-encoding nucleic acid molecule encodes a differentsuperantigen than a second superantigen-encoding nucleic acid molecule.

One or more recombinant molecules of the present invention can be usedto produce an encoded product (i.e., a superantigen protein, a cytokineand a chemokine protein) of the present invention. In one embodiment, anencoded product of the present invention is produced by expressing anucleic acid molecule of the present invention in a cell underconditions effective to produce the protein. A preferred method toproduce an encoded protein is by transforming (i.e., introducing arecombinant molecule into a cell in such a manner that the recombinantmolecule is expressed by the cell) a host cell with one or morerecombinant molecules of the present invention to form a recombinantcell. Suitable host cells to transform include any cell into which arecombinant molecule can be introduced. Host cells can be eitheruntransformed cells or cells that are already transformed with at leastone nucleic acid molecule. Host cells of the present invention can beany cell capable of producing a superantigen, a cytokine and/or achemokine of the present invention, including bacterial, fungal, animalparasite, insect and animal cells. A preferred host cell includes amammalian and a bird cell. A more preferred host cell includes mammalianlymphocytes, muscle cells, hematopoietic precursor cells, mast cells,natural killer cells, macrophages, monocytes, epithelial cells,endothelial cells, dendritic cells, mesenchymal cells, Langerhans cells,cells found in granulomas and tumor cells of any cellular origin. Aneven more preferred host cell of the present invention includesmammalian mesenchymal cells, epithelial cells, endothelial cells,macrophages, monocytes, muscle cells, T cells and dendritic cells.

According to the present invention, a recombinant molecule can beintroduced into a host cell in vivo (i.e., in an animal) or in vitro(i.e., outside of an animal, such as in tissue culture). Introduction ofa nucleic acid molecule into a host cell can be accomplished by anymethod by which a nucleic acid molecule can be inserted into the cell.Transformation techniques include, but are not limited to, transfection,electroporation, microinjection, lipofection, adsorption, and protoplastfusion. Preferred methods to introduce a recombinant molecule into hostcells in vivo include lipofection and adsorption (discussed in detailbelow).

A recombinant cell of the present invention comprises a cell into whicha nucleic acid molecule that encodes a superantigen, a cytokine and/or achemokine has been introduced. In one embodiment, a recombinant cell ofthe present invention is transformed with a nucleic acid molecule thatincludes at least a portion of PCR₃ -SEB, PCR₃ -SEA, PCR₃ -SEB.S, PCR₃-SEA.S, PCR₃ -TSST, or combinations thereof. Particularly preferredrecombinant cells include cells transformed with PCR₃ -SEB, PCR₃ -SEA,PCR₃ -SEB.S, PCR₃ -SEA.S or PCR₃ -TSST, with PCR₃ -SEB.S, PCR₃ -SEA.S orPCR₃ -TSST being even more preferred.

In another embodiment, a recombinant cell of the present invention istransformed with a nucleic acid molecule that includes at least aportion of PCR₃ -SEB, PCR₃ -SEA, PCR₃ -SEB.S, PCR₃ -SEA.S, PCR₃ -TSST orcombinations thereof, and PCR₃ -GM₃. Particularly preferred stimulatoryrecombinant cells include cells transformed with PCR₃ -SEA and PCR₃-GM₃, PCR₃ -SEA.S and PCR₃ -GM₃, PCR₃ -SEB and PCR₃ -GM₃, PCR₃ -SEB.Sand PCR₃ -GM₃, or PCR₃ -TSST and PCR₃ -GM₃. Even more preferredstimulatory recombinant cells include cells transformed with PCR₃ -SEB.Sand PCR₃ -GM₃, or PCR₃ -SEA.S and PCR₃ -GM₃, and PCR₃ -TSST and PCR₃-GM₃.

Recombinant DNA technologies can be used to improve expression oftransformed nucleic acid molecules by manipulating, for example, thenumber of copies of the nucleic acid molecules within a host cell, theefficiency with which those nucleic acid molecules are transcribed, theefficiency with which the resultant transcripts are translated, and theefficiency of post-translational modifications. Recombinant techniquesuseful for increasing the expression of nucleic acid molecules of thepresent invention include, but are not limited to, operatively linkingnucleic acid molecules to high-copy number plasmids, integration of thenucleic acid molecules into one or more host cell chromosomes, additionof vector stability sequences to plasmids, substitutions ormodifications of transcription control signals (e.g., promoters,operators, enhancers), substitutions or modifications of translationalcontrol signals (e.g., ribosome binding sites, Shine-Dalgarnosequences), modification of nucleic acid molecules of the presentinvention to correspond to the codon usage of the host cell, anddeletion of sequences that destabilize transcripts. The activity of anexpressed recombinant protein of the present invention may be improvedby fragmenting, modifying, or derivatizing nucleic acid moleculesencoding such a protein.

In another embodiment of the present invention, a therapeuticcomposition further comprises a pharmaceutically acceptable carrier. Asused herein, a "carrier" refers to any substance suitable as a vehiclefor delivering a nucleic acid molecule of the present invention to asuitable in vivo or in vitro site. As such, carriers can act as apharmaceutically acceptable excipient of a therapeutic compositioncontaining a nucleic acid molecule of the present invention. Preferredcarriers are capable of maintaining a nucleic acid molecule of thepresent invention in a form that, upon arrival of the nucleic acidmolecule to a cell, the nucleic acid molecule is capable of entering thecell and being expressed by the cell. Carriers of the present inventioninclude: (1) excipients or formularies that transport, but do notspecifically target a nucleic acid molecule to a cell (referred toherein as non-targeting carriers); and (2) excipients or formulariesthat deliver a nucleic acid molecule to a specific site in an animal ora specific cell (i.e., targeting carriers). Examples of non-targetingcarriers include, but are not limited to water, phosphate bufferedsaline, Ringer's solution, dextrose solution, serum-containingsolutions, Hank's solution, other aqueous physiologically balancedsolutions, oils, esters and glycols. Aqueous carriers can containsuitable auxiliary substances required to approximate the physiologicalconditions of the recipient, for example, by enhancing chemicalstability and isotonicity.

Suitable auxiliary substances include, for example, sodium acetate,sodium chloride, sodium lactate, potassium chloride, calcium chloride,and other substances used to produce phosphate buffer, Tris buffer, andbicarbonate buffer. Auxiliary substances can also include preservatives,such as thimerosal, m- and o-cresol, formalin and benzol alcohol.Preferred auxiliary substances for aerosol delivery include surfactantsubstances non-toxic to an animal, for example, esters or partial estersof fatty acids containing from about six to about twenty-two carbonatoms. Examples of esters include, caproic, octanoic, lauric, palmitic,stearic, linoleic, linolenic, olesteric, and oleic acids. Other carrierscan include metal particles (e.g., gold particles) for use with, forexample, a biolistic gun through the skin. Therapeutic compositions ofthe present invention can be sterilized by conventional methods and/orlyophilized.

Targeting carriers are herein referred to as "delivery vehicles."Delivery vehicles of the present invention are capable of delivering atherapeutic composition of the present invention to a target site in ananimal. A "target site" refers to a site in an animal to which onedesires to deliver a therapeutic composition. For example, a target sitecan be a malignant tumor cell, a non-malignant tumor cell, a lymph nodeor a lesion caused by an infectious agent, or an area around such cell,tumor or lesion, which is targeted by direct injection or delivery usingliposomes or other delivery vehicles. Examples of delivery vehiclesinclude, but are not limited to, artificial and natural lipid-containingdelivery vehicles. Natural lipid-containing delivery vehicles includecells and cellular membranes. Artificial lipid-containing deliveryvehicles include liposomes and micelles. A delivery vehicle of thepresent invention can be modified to target to a particular site in ananimal, thereby targeting and making use of a nucleic acid molecule ofthe present invention at that site. Suitable modifications includemanipulating the chemical formula of the lipid portion of the deliveryvehicle and/or introducing into the vehicle a compound capable ofspecifically targeting a delivery vehicle to a preferred site, forexample, a preferred cell type. Specifically targeting refers to causinga delivery vehicle to bind to a particular cell by the interaction ofthe compound in the vehicle to a molecule on the surface of the cell.Suitable targeting compounds include ligands capable of selectively(i.e., specifically) binding another molecule at a particular site.Examples of such ligands include antibodies, antigens, receptors andreceptor ligands. For example, an antibody specific for an antigen foundon the surface of a cancer cell can be introduced to the outer surfaceof a liposome delivery vehicle so as to target the delivery vehicle tothe cancer cell. Tumor cell ligands include ligands capable of bindingto a molecule on the surface of a tumor cell. Manipulating the chemicalformula of the lipid portion of the delivery vehicle can modulate theextracellular or intracellular targeting of the delivery vehicle. Forexample, a chemical can be added to the lipid formula of a liposome thatalters the charge of the lipid bilayer of the liposome so that theliposome fuses with particular cells having particular chargecharacteristics.

A preferred delivery vehicle of the present invention is a liposome. Aliposome is capable of remaining stable in an animal for a sufficientamount of time to deliver a nucleic acid molecule of the presentinvention to a preferred site in the animal. A liposome of the presentinvention is preferably stable in the animal into which it has beenadministered for at least about 30 minutes, more preferably for at leastabout 1 hour and even more preferably for at least about 24 hours.

A liposome of the present invention comprises a lipid composition thatis capable of targeting a nucleic acid molecule of the present inventionto a particular, or selected, site in an animal. Preferably, the lipidcomposition of the liposome is capable of targeting to any organ of ananimal, more preferably to the lung, liver, spleen, heart brain, lymphnodes and skin of an animal, and even more preferably to the lung of ananimal.

A liposome of the present invention comprises a lipid composition thatis capable of fusing with the plasma membrane of the targeted cell todeliver a nucleic acid molecule into a cell. Preferably, thetransfection efficiency of a liposome of the present invention is atleast about 0.5 microgram (μg) of DNA per 16 nanomole (nmol) of liposomedelivered to about 10⁶ cells, more preferably at least about 1.0 μg ofDNA per 16 nmol of liposome delivered to about 10⁶ cells, and even morepreferably at least about 2.0 μg of DNA per 16 nmol of liposomedelivered to about 10⁶ cells.

A preferred liposome of the present invention is between about 100 andabout 500 nanometers (nm), more preferably between about 150 and about450 nm and even more preferably between about 200 and about 400 nm indiameter.

Suitable liposomes for use with the present invention include anyliposome. Preferred liposomes of the present invention include thoseliposomes standardly used in, for example, gene delivery methods knownto those of skill in the art. More preferred liposomes compriseliposomes having a polycationic lipid composition and/or liposomeshaving a cholesterol backbone conjugated to polyethylene glycol. Evenmore preferred liposomes include liposomes produced according to themethod described in Example 2.

In one embodiment, a liposome of the present invention comprises acompound capable of targeting the liposome to a tumor cell. Such aliposome preferably includes a tumor cell ligand exposed on the outersurface of the liposome.

Complexing a liposome with a nucleic acid molecule of the presentinvention can be achieved using methods standard in the art (see, forexample, methods described in Example 2). A suitable concentration of anucleic acid molecule of the present invention to add to a liposomeincludes a concentration effective for delivering a sufficient amount ofnucleic acid molecule to a cell such that the cell can producesufficient superantigen and/or cytokine protein to regulate effectorcell immunity in a desired manner. Preferably, nucleic acid moleculesare combined with liposomes at a ratio of from about 0.1 μg to about 10μg of nucleic acid molecule of the present invention per about 8 nmolliposomes, more preferably from about 0.5 μg to about 5 μg of nucleicacid molecule per about 8 nmol liposomes, and even more preferably about1.0 μg of nucleic acid molecule per about 8 nmol liposomes.

Another preferred delivery vehicle comprises a recombinant virusparticle vaccine. A recombinant virus particle vaccine of the presentinvention includes a therapeutic composition of the present invention,in which the recombinant molecules contained in the composition arepackaged in a viral coat that allows entrance of DNA into a cell so thatthe DNA is expressed in the cell. A number of recombinant virusparticles can be used, including, but not limited to, those based onalphaviruses, poxviruses, adenoviruses, herpesviruses, arena virus andretroviruses.

Another preferred delivery vehicle comprises a recombinant cell vaccine.Preferred recombinant cell vaccines of the present invention includetumor vaccines, in which allogeneic (i.e., cells derived from a sourceother than a patient, but that are histiotype compatible with thepatient) or autologous (i.e., cells isolated from a patient) tumor cellsare transfected with recombinant molecules contained in a therapeuticcomposition, irradiated and administered to a patient by, for example,intradermal, intravenous or subcutaneous injection. Therapeuticcompositions to be administered by tumor cell vaccine, includerecombinant molecules of the present invention without carrier. Tumorcell vaccine treatment is useful for the treatment of both tumor andmetastatic cancer. Use of a tumor vaccine of the present invention isparticular useful for treating metastatic cancer, including preventingmetastatic disease, as well as, curing existing metastatic disease.Methods for developing and administering include those standard in theart (see for example, Dranoff et al., Proc. Natl. Acad. Sci. USA90:3539-3543, 1993, which is incorporated herein by reference in itsentirety).

A therapeutic composition of the present invention is useful for thetreatment of a variety of diseases, including, but not limited to,cancer, autoimmune disease, infectious diseases, and other diseases thatcan be alleviated by either stimulating or suppressing T cell activity.As used herein, the term "treatment" refers to protecting an animal froma disease or alleviating a disease in an animal. A therapeuticcomposition of the present invention is advantageous for the treatmentof cancer in that the composition overcomes the mechanisms by whichcancer cells avoid immune elimination (i.e., by which cancer cells avoidthe immune response effected by the animal in response to the disease).Cancer cells can avoid immune elimination by, for example, being onlyslightly immunogenic, modulating cell surface antigens and inducingimmune suppression. Suitable therapeutic compositions for use in thetreatment of cancer comprises a superantigen-encoding recombinantmolecule; or a combination of a superantigen-encoding recombinantmolecule, with a cytokine-encoding recombinant molecule and/or achemokine recombinant molecule of the present invention. Preferredtherapeutic compositions for use in the treatment of cancer comprises asuperantigen-encoding recombinant molecule; or a combination of asuperantigen-encoding recombinant molecule with a cytokine-encodingrecombinant molecule and/or a chemokine recombinant molecule of thepresent invention combined (separately or together) with a deliveryvehicle, preferably a liposome, such as disclosed herein. A therapeuticcomposition of the present invention, upon entering targeted cells,leads to the production of superantigen, cytokine and/or chemokineprotein that activate cytotoxic T cells, natural killer cells, T helpercells and macrophages. Such cellular activation overcomes the otherwiserelative lack of immune response to cancer cells, leading to thedestruction of such cells.

A therapeutic composition of the present invention is useful for thetreatment of cancers, both tumors and metastatic forms of cancer.Treatment with the therapeutic composition overcomes the disadvantagesof traditional treatments for metastatic cancers. For example,compositions of the present invention can target dispersed metastaticcancer cells that cannot be treated using surgical methods. In addition,administration of such compositions do not result in the harmful sideeffects caused by chemotherapy and radiation therapy.

A therapeutic composition of the present invention is preferably used totreat cancers, including, but not limited to, melanomas, squamous cellcarcinoma, breast cancers, head and neck carcinomas, thyroid carcinomas,soft tissue sarcomas, bone sarcomas, testicular cancers, prostaticcancers, ovarian cancers, bladder cancers, skin cancers, brain cancers,angiosarcomas, hemangiosarcomas, mast cell tumors, primary hepaticcancers, lung cancers, pancreatic cancers, gastrointestinal cancers,renal cell carcinomas, hematopoietic neoplasias, leukemias andlymphomas. Particularly preferred cancers to treat with a therapeuticcomposition of the present invention, include melanomas, lung cancers,thyroid carcinomas, breast cancers, renal cell carcinomas, squamous cellcarcinomas, brain tumors and skin cancers. A therapeutic composition ofthe present invention is useful for treating tumors that can form insuch cancers, including malignant and benign tumors.

A therapeutic composition of the present invention is also advantageousfor the treatment of infectious diseases as a long term, targetedtherapy for primary lesions (e.g., granulomas) resulting from thepropagation of a pathogen. As used herein, the term "lesion" refers to alesion formed by infection of an animal with a pathogen. Preferredtherapeutic compositions for use in the treatment of an infectiousdisease comprise a superantigen-encoding recombinant molecule; or acombination of a superantigen-encoding recombinant molecule, with acytokine-encoding recombinant molecule and/or a chemokine recombinantmolecule of the present invention. More preferred therapeuticcompositions for use in the treatment of infectious disease comprise asuperantigen-encoding recombinant molecule; or a combination ofsuperantigen-encoding recombinant molecule, with a cytokine-encodingrecombinant molecule and/or a chemokine recombinant molecule of thepresent invention combined with a delivery vehicle, preferably aliposome of the present invention. Similar to the mechanism describedfor the treatment of cancer, treatment of infectious diseases withsuperantigen, cytokine and/or chemokine can result in increased T cell,natural killer cell, and macrophage cell activity that overcome therelative lack of immune response to a lesion formed by a pathogen.

A therapeutic composition of the present invention is particularlyuseful for the treatment of infectious diseases caused by pathogens,including, but not limited to, intracellular bacteria (i.e., a bacteriathat resides in a host cell), internal parasites, pathogenic fungi andendoparasites. Particularly preferred infectious diseases to treat witha therapeutic composition of the present invention include tuberculosis,leprosy, aspergillosis, coccidioidomycosis, cryptococcoses,leishmaniasis and toxoplasmosis.

In order to treat an animal with disease, a therapeutic composition ofthe present invention is administered to the animal in an effectivemanner such that the composition is capable of treating that animal fromdisease. For example, a recombinant molecule, when administered to ananimal in an effective manner, is able to stimulate effector cellimmunity in a manner that is sufficient to alleviate the diseaseafflicting the animal. According to the present invention, treatment ofa disease refers to alleviating a disease and/or preventing thedevelopment of a secondary disease resulting from the occurrence of aprimary disease.

An effective administration protocol (i.e., administering a therapeuticcomposition in an effective manner) comprises suitable dose parametersand modes of administration that result in treatment of a disease.Effective dose parameters and modes of administration can be determinedusing methods standard in the art for a particular disease. Such methodsinclude, for example, determination of survival rates, side effects(i.e., toxicity) and progression or regression of disease. Inparticular, the effectiveness of dose parameters and modes ofadministration of a therapeutic composition of the present inventionwhen treating cancer can be determined by assessing response rates. Suchresponse rates refer to the percentage of treated patients in apopulation of patients that respond with either partial or completeremission. Remission can be determined by, for example, measuring tumorsize or microscopic examination for the presence of cancer cells in atissue sample.

In accordance with the present invention, a suitable single dose size isa dose that is capable of treating an animal with disease whenadministered one or more times over a suitable time period. Doses canvary depending upon the disease being treated. In the treatment ofcancer, a suitable single dose can be dependent upon whether the cancerbeing treated is a primary tumor or a metastatic form of cancer. Dosesof a therapeutic composition of the present invention suitable for usewith direct injection techniques can be used by one of skill in the artto determine appropriate single dose sizes for systemic administrationbased on the size of an animal. A suitable single dose of a therapeuticcomposition to treat a tumor is a sufficient amount of asuperantigen-encoding recombinant molecule; or a superantigen-encodingrecombinant molecule, with a cytokine-encoding recombinant moleculeand/or a chemokine recombinant molecule to reduce, and preferablyeliminate, the tumor following transfection of the recombinant moleculesinto cells at or near the tumor site. A preferred single dose of thesuperantigen-encoding recombinant molecule is an amount that, whentransfected into a target cell population, leads to the production offrom about 250 femtograms (fg) to about 1 μg, preferably from about 500fg to about 500 picogram (pg), and more preferably from about 1 pg toabout 100 pg of superantigen per transfected cell. A preferred singledose of a cytokine-encoding recombinant molecule is an amount that, whentransfected into a target cell population, leads to the production offrom about 10 pg to about 1 μg, preferably from about 100 pg to about750 pg, and more preferably about 500 pg of cytokine per transfectant. Apreferred single dose of a chemokine-encoding recombinant molecule is anamount that, when transfected into a target cell population, leads tothe production of from about 1 fg to about 1 μg, preferably from about 1pg to about 10 ng, and more preferably from about 1 pg to about 1 ngchemokine per transfectant.

A suitable single dose of a superantigen-encoding recombinant molecule;or a combination of a superantigen-encoding recombinant molecule, with acytokine-encoding recombinant molecule and/or a chemokine-encodingrecombinant molecule in a non-targeting carrier to administer to ananimal to treat a tumor, is an amount capable of reducing, andpreferably eliminating, the tumor following transfection of therecombinant molecules into cells at or near the tumor site. A preferredsingle dose of a therapeutic composition to treat a tumor is from about100 μg to about 2 milligrams (mg) of total recombinant molecules, morepreferably from about 150 μg to about 1 mg of total recombinantmolecules, and even more preferably from about 200 μg to about 800 μg oftotal recombinant molecules. A preferred single dose of asuperantigen-encoding recombinant molecule complexed with liposomes, isfrom about 100 μg of total DNA per 800 nmol of liposome to about 2 mg oftotal recombinant molecules per 16 micromole (μmol) of liposome, morepreferably from about 150 μg per 1.2 μmol of liposome to about 1 mg oftotal recombinant molecules per 8 μmol of liposome, and even morepreferably from about 200 μg per 2 μmol of liposome to about 400 μg oftotal recombinant molecules per 3.2 μmol of liposome.

A preferred single dose of a cytokine-encoding recombinant molecule or achemokine-encoding recombinant molecule in a non-targeting carrier toadminister to an animal to treat a tumor, is from about 100 μg to about2 mg of total recombinant molecules, more preferably from about 150 μgto about 1 mg of total recombinant molecules, and even more preferablyfrom about 200 μg to about 400 μg of total recombinant molecules. Apreferred single dose of a cytokine-encoding recombinant molecule or achemokine-encoding recombinant molecule complexed with liposomes toadminister to an animal to treat a tumor, is from about 100 μg of totalrecombinant molecules per 800 nmol of liposome to about 2 mg of totalrecombinant molecules per 16 μmol of liposome, more preferably fromabout 150 μg per 1.2 μmol of liposome to about 1 mg of total recombinantmolecules per 8 μmol of liposome, and even more preferably from about200 μg per 2 μmol of liposome to about 400 μg of total recombinantmolecules per 6.4 μmol of liposome.

A preferred single dose of a superantigen-encoding recombinant moleculein a non-targeting carrier to administer to an animal treat a metastaticcancer, is from about 100 μg to about 4 mg of total recombinantmolecules, more preferably from about 150 μg to about 3 mg of totalrecombinant molecules, and even more preferably from about 200 μg toabout 2 mg of total recombinant molecules. A preferred single dose of asuperantigen-encoding recombinant molecule complexed with liposomes toadminister to an animal to treat a metastatic cancer, is from about 100μg of total recombinant molecules per 800 nmol of liposome to about 4 mgof total recombinant molecules per 32 μmol of liposome, more preferablyfrom about 200 μg per 1.6 μm of liposome to about 3 mg of totalrecombinant molecules per 24 μmol of liposome, and even more preferablyfrom about 400 μg per 3.2 μmol of liposome to about 2 mg of totalrecombinant molecules per 16 μmol of liposome.

A preferred single dose of a cytokine-encoding recombinant molecule or achemokine-encoding recombinant molecule in a non-targeting carrier toadminister to an animal to treat a metastatic cancer, is from about 100μg to about 4.0 mg of total recombinant molecules, more preferably fromabout 150 μg to about 3 mg of total recombinant molecules, and even morepreferably from about 200 μg to about 2 mg of total recombinantmolecules. A preferred single dose of a cytokine-encoding recombinantmolecule or a chemokine-encoding recombinant molecule complexed withliposomes to administer to an animal to treat a metastatic cancer, isfrom about 100 μg of total recombinant molecules per 800 nmol ofliposome to about 4.0 mg of total recombinant molecules per 32 μmol ofliposome, more preferably from about 200 μg per 1.6 μmol of liposome toabout 3 mg of total recombinant molecules per 24 μmol of liposome, andeven more preferably from about 400 μg per 3.2 μmol of liposome to about2 μg of total recombinant molecules per 16 μmol of liposome.

According to the present invention, a single dose of a therapeuticcomposition useful to treat a lesion, comprising a superantigen-encodingrecombinant molecule in a non-targeting carrier or liposomes,respectively, and a cytokine-encoding recombinant molecule in anon-targeting carrier or liposomes, respectively, is substantiallysimilar to those doses used to treat a tumor (as described in detailabove).

The number of doses administered to an animal is dependent upon theextent of the disease and the response of an individual patient to thetreatment. For example, a large tumor may require more doses than asmaller tumor. In some cases, however, a patient having a large tumormay require fewer doses than a patient with a smaller tumor, if thepatient with the large tumor responds more favorably to the therapeuticcomposition than the patient with the smaller tumor. Thus, it is withinthe scope of the present invention that a suitable number of dosesincludes any number required to cause regression of a disease. Apreferred protocol is monthly administrations of single doses (asdescribed above) for up to about 1 year. A preferred number of doses ofa therapeutic composition comprising a superantigen-encoding recombinantmolecule; or a combination of a superantigen-encoding recombinantmolecule, with a cytokine-encoding recombinant molecule and/or achemokine-encoding recombinant molecule in a non-targeting carrier orcomplexed with liposomes in order to treat a tumor is from about 1 toabout 10 administrations per patient, preferably from about 2 to about 8administrations per patient, and even more preferably from about 3 toabout 5 administrations per patient. Preferably, such administrationsare given once every 2 weeks until signs of remission appear, then oncea month until the disease is gone.

A preferred number of doses of a therapeutic composition comprising asuperantigen-encoding recombinant molecule; or a combination of asuperantigen-encoding recombinant molecule, with a cytokine-encodingrecombinant molecule and/or a chemokine-encoding recombinant molecule ina non-targeting carrier or completed with liposomes in order to treat ametastatic cancer, is from about 2 to about 10 administrations patient,more preferably from about 3 to about 8 administrations per patient, andeven more preferably from about 3 to about 7 administrations perpatient. Preferably, such administrations are given once every 2 weeksuntil signs of remission appear, then once a month until the disease isgone.

According to the present invention, the number of doses of a therapeuticcomposition to treat a lesion comprising a superantigen-encodingrecombinant molecule; or a combination of a superantigen-encodingrecombinant molecule, with a cytokine-encoding recombinant moleculeand/or a chemokine-encoding recombinant molecule, in a non-targetingcarrier or liposomes, respectively, is substantially similar to thosenumber of doses used to treat a tumor (as described in detail above).

A therapeutic composition is administered to an animal in a fashion toenable expression of an introduced recombinant molecule of the presentinvention into a curative protein in the animal to be treated fordisease. A therapeutic composition can be administered to an animal in avariety of methods including, but not limited to, local administrationof the composition into a site in an animal. Examples of such sitesinclude lymph nodes, a site that contains abnormal cells or pathogens tobe destroyed (e.g., injection locally within the area of a tumor or alesion); and systemic administration.

Therapeutic compositions to be delivered by local administrationinclude: (a) recombinant molecules of the present invention in anon-targeting carrier (e.g., as "naked" DNA molecules, such as istaught, for example in Wolff et al., 1990, Science 247, 1465-1468); and(b) recombinant molecules of the present invention complexed to adelivery vehicle of the present invention. Suitable delivery vehiclesfor local administration comprise liposomes. Delivery vehicles for localadministration can further comprise ligands for targeting the vehicle toa particular site (as described in detail herein).

A preferred method of local administration is by direct injection.Direct injection techniques are particularly useful for the treatment ofdisease by, for example, injecting the composition into a mass formed byabnormal cells, a lymph node or a granuloma mass induced by pathogens.Preferably, a recombinant molecule of the present invention complexedwith a delivery vehicle is administered by direct injection into orlocally within the area of a tumor mass, a lymph node, a granuloma massor a cancer cell. Administration of a composition locally within an areaof a mass or a cell refers to injecting the composition centimeters andpreferably, millimeters within the mass or the cell. A preferred tumormass to inject includes discrete inner body and cutaneous solid tumors.A preferred inner body tumor to inject includes a discrete solid tumorthat forms in the brain, breast, liver, kidney, colon, prostate,testicular, ovary, spleen and/or lymph node. A preferred cutaneous tumorto inject includes a discrete solid melanoma.

A preferred lymph node to inject includes a draining lymph node that"drains" a site containing abnormal cells or pathogens. As used herein,the term "draining lymph node" refers to a lymph node that is locateddownstream of a site containing abnormal cells or pathogens is based onthe direction of the lymphatic flow of an animal (see general discussionin Hole, Human Anatomy and Physiology, Edward G. Jaffe, ed., Wm. C BrownPublishers, Dubuque, Iowa; and G. C. Christiansen et al., Anatomy of theDog, W. B. Saunders Publishers, Philadelphia, Pa., 1979; both of whichare incorporated herein by this reference). A preferred draining lymphnode to inject comprises the draining lymph node most proximal to a sitecontaining abnormal cells or pathogens. Thus, a skilled artisan canchoose the site of lymph node injection based upon the location of thesite containing abnormal cells or pathogens. Examples of lymph nodes toinjection include: the mandibular lymph node if a tumor is located inthe oral cavity; and the superficial cervical lymph node of a tumor islocated in the front leg region. Effector cells travel from a sitecontaining abnormal cells or pathogens. Injection of a therapeuticcomposition of the present invention into a lymph node can result inexpression of a superantigen, a cytokine and/or a chemokine by aneffector cell from the lymph node or that has drained into the lymphnode. Such expression can result in the activation of T lymphocytes,which can travel back to the site containing abnormal cells or pathogensto enhance the immune response at the site.

Another method of local administration is to contact a therapeuticcomposition of the present invention in or around a surgical wound. Forexample, a patient can undergo surgery to remove a tumor. Upon removalof the tumor, the therapeutic composition can be coated on the surfaceof tissue inside the wound or the composition can be injected into areasof tissue inside the wound. Such local administration is useful fortreating cancer cells not excised by the surgical procedure, as well as,preventing recurrence of the primary tumor or development of a secondarytumor in the area of the surgery.

In one embodiment, a therapeutic composition of the present inventioncan be introduced to a tumor cell in vivo. In another embodiment, atherapeutic composition of the present invention can be introduced to anon-tumor cell in vivo or in vitro. Methods to introduce a therapeuticcomposition in vivo are disclosed herein. Methods to introduce atherapeutic composition in vitro include methods standard in the art,such as culturing cells in the presence of a therapeutic composition fora sufficient amount of time to enable a nucleic acid molecule of thepresent invention to pass through the plasma membrane in a cell andsubsequently to be expressed in the cell.

Therapeutic compositions useful in systemic administration, includerecombinant molecules of the present invention complexed to a targeteddelivery vehicle of the present invention. Suitable delivery vehiclesfor use with systemic administration comprise liposomes comprisingligands for targeting the vehicle to a particular site, preferablyligands for targeting the vehicle to a site of a cancer or a lesion(depending upon the disease being treated). For cancer treatment,ligands capable of selectively binding to a cancer cell or to a cellwithin the area of a cancer cell are preferred. Systemic administrationis useful for the treatment of both tumor and metastatic cancer andsystemic infectious diseases. Systemic administration is particularlyuseful for the treatment of metastatic forms of cancer, in which thecancer cells are dispersed (i.e., not localized within a single tumormass). Systemic administration is particularly advantageous when organs,in particular difficult to reach organs (e.g., heart, spleen, lung orliver) are the targeted sites of treatment.

Preferred methods of systemic administration, include intravenousinjection, aerosol, oral and percutaneous (topical) delivery.Intravenous injections can be performed using methods standard in theart. Aerosol delivery can also be performed using methods standard inthe art (see, for example, Stribling et al., Proc. Natl. Acad. Sci. USA189:11277-11281, 1992, which is incorporated herein by reference in itsentirety). Oral delivery can be performed by complexing a therapeuticcomposition of the present invention to a carrier capable ofwithstanding degradation by digestive enzymes in the gut of an animal.Examples of such carriers, include plastic capsules or tablets, such asthose known in the art. Topical delivery can be performed by mixing atherapeutic composition of the present invention with a lipophilicreagent (e.g., DMSO) that is capable of passing into the skin.

Therapeutic compositions of the present invention can be administered toany animal, preferably to mammals and birds, and more preferably tohumans, house pets, economic produce animals and zoo animals. Economicproduce animals include animals to be consumed or that produce usefulproducts (e.g., sheep for wool production). Zoo animals include thoseanimals harbored in zoos. Preferred animals to protect include humans,dogs, cats, sheep, cattle, horses and pigs, with humans and dogs beingparticularly preferred. While a therapeutic composition of the presentinvention is effective to treat disease in inbred species of animals,the composition is particularly useful for treating outbred species ofanimals, in particular those having tumors.

Yet another embodiment of the present invention is a method to suppressT cell activity in an animal, the method comprising administering to ananimal an effective amount of a therapeutic composition comprising: (a)a naked nucleic acid molecule encoding a superantigen; and (b) apharmaceutically acceptable carrier, in which the nucleic acid moleculeis operatively linked to a transcription control sequence, and in whichthe therapeutic composition is targeted to a site in the animal thatcontains excessive T cell activity.

Suitable embodiments, single dose sizes, number of doses and modes ofadministration of a therapeutic composition of the present inventionuseful in a treatment method of the present invention are disclosed indetail herein.

A therapeutic composition of the present invention is also advantageousfor the treatment of autoimmune diseases in that the compositionsuppresses the harmful stimulation of T cells by autoantigens (i.e., a"self", rather than a foreign antigen). Superantigen-encodingrecombinant molecules in a therapeutic composition, upon transfectioninto a cell, produce superantigens that delete harmful populations of Tcells involved in an autoimmune disease. A preferred therapeuticcomposition for use in the treatment of autoimmune disease comprises asuperantigen-encoding recombinant molecule of the present invention. Amore preferred therapeutic composition for use in the treatment ofautoimmune disease comprises a superantigen-encoding recombinantmolecule combined with a non-targeting carrier of the present invention,preferably saline or phosphate buffered saline.

Such a therapeutic composition of the present invention is particularlyuseful for the treatment of autoimmune diseases, including but notlimited to, multiple sclerosis, systemic lupus erythematosus, myastheniagravis, rheumatoid arthritis, insulin dependent diabetes mellitus,psoriasis, polyarteritis, immune mediated vasculitides, immune mediatedglomerulonephritis, inflammatory neuropathies and sarcoidosis.

A single dose of a superantigen-encoding nucleic acid molecule in anon-targeting carrier to administer to an animal to treat an autoimmunedisease is from about 0.1 μg to about 200 μg of total recombinantmolecules per kilogram (kg) of body weight, more preferably from about0.5 μg to about 150 μg of total recombinant molecules per kg of bodyweight, and even more preferably from about 1 μg to about 10 μg of totalrecombinant molecules per kg of body weight.

The number of doses of a superantigen-encoding recombinant molecule in anon-targeting carrier to be administered to an animal to treat anautoimmune disease is an injection about once every 6 months, morepreferably about once every 3 months, and even more preferably aboutonce a month.

A preferred method to administer a therapeutic composition of thepresent invention to treat an autoimmune disease is by localadministration, preferably direct injection. Direct injection techniquesare particularly important in the treatment of an autoimmune disease.Preferably, a therapeutic composition is injected directly into musclecells in a patient, which results in prolonged expression (e.g., weeksto months) of a recombinant molecule of the present invention.Preferably, a recombinant molecule of the present invention in the formof "naked DNA" is administered by direct injection into muscle cells ina patient.

Another aspect of the present invention is an adjuvant for use with anucleic acid-based vaccine to protect an animal from a disease or aremedy to treat a diseased animal. Adjuvants of the present inventioncomprise: (a) a superantigen-encoding nucleic acid molecule of thepresent invention; or (b) a combination of a superantigen-encodingnucleic acid molecule of the present invention with a cytokine nucleicacid molecule of the present invention, a chemokine nucleic acidmolecule of the present invention or mixtures thereof.

Suitable compounds to combine with an adjuvant of the present invention,to form an adjuvant composition (i.e., a vaccine composition useful as apreventative therapeutic reagent or a therapeutic remedy useful toalleviate a disease) of the present invention, include any compound thatis administered to an animal as an immunogen. As used herein, animmunogen of the present invention comprises a compound capable ofeliciting an immune response in an animal. Preferably, an immunogen ofthe present invention is derived from a foreign agent including apathogen. Also preferably, an immunogen of the present inventionincludes an allergen (organic or inorganic), tumor antigens andself-antigens.

A preferred immunogen is derived from a pathogen including, but notlimited to, a virus, a bacteria, a eukaryotic parasite and unicellularprotozoa (e.g., amoeba). Preferred eukaryotic parasites includeprotozoan parasites, helminth parasites (such as nematodes, cestodes,trematodes, ectoparasites and fungi.

A preferred immunogen also includes an allergen including, but notlimited to, a plant allergen, an animal allergen, a bacterial allergen,a parasitic allergen, a metal-based allergen that causes contactsensitivity and inorganic allergens such as silica, beryllium,xenobiotics, synthetic drugs and dyes. A more preferred allergenincludes weed, grass, tree, peanut, mite, flea, cat, house dust andbacterial products antigens.

A preferred immunogen derived from a bacteria includes an immunogen thatprotects an animal from or alleviates Mycobacterium infection, inparticular M. tuberculosis, M. leprae, M. avium, and/or M. bovisinfection. A more preferred bacterial immunogen of the present inventionincludes a peptide, mimetopes thereof and compositions containing thesame, as disclosed in U.S. patent Ser. No. 08/484,169, filed Jun. 7,1995, which is incorporated herein by this reference.

In one embodiment, an immunogen comprises a nucleic acid moleculeencoding an immunogenic protein. Such immunogen-encoding nucleic acidmolecules can be designed by those of skill in the art based upon theamino acid sequence of the immunogen. In addition, a recombinantmolecule encoding an immunogen of the present invention can be producedusing the recombinant DNA technology disclosed herein and known to thoseof skill in the art. In other embodiments, an immunogen can comprise apeptide, a polypeptide or a chemical compound as disclosed herein. Allsuch embodiments of an immunogen are useful with an adjuvant of thepresent invention.

In order to treat an animal (i.e., vaccinate or remedy), an adjuvantcomposition of the present invention is administered to the animal in aneffective manner such that the composition is capable of protecting ananimal from or alleviating a disease. For example, an adjuvant, whenadministered to an animal in an effective manner, is able to stimulateeffector cell immunity in a manner that is sufficient to prevent aninitial or continued disease response by the subject animal.

An effective administration protocol (i.e., administering an adjuvantcomposition in an effective manner) comprises suitable dose parameters,and modes and times of administration that result in the treatment of ananimal. Effective dose parameters and modes of administration can bedetermined using methods standard in the art for a particular adjuvantcomposition. Such methods include, for example: determination of sideeffects (i.e., toxicity) of an adjuvant composition; progression of adisease upon administration of an adjuvant composition; magnitude and/orduration of antibody response by an animal against an immunogencontained in an adjuvant composition; magnitude and/or duration of acell mediated immune response in an animal against an adjuvantcomposition; similarity of an immune response to an adjuvant compositionin different species of animals; and/or effect of breed (in non-humananimals) or race (in humans) on responsiveness to an adjuvantcomposition. In particular, the effectiveness of dose parameters andmodes of administration of an adjuvant composition of the presentinvention can be determined by assessing antibody production in vivo,skin test sensitivities in vivo, cytokine production in vitro,antigen-specific proliferation in vitro, cytotoxic T cell activity invitro, reduction of tumor burden in vivo and/or reduction of infectiousagent burden in vivo. Tests standard in the art can be used to determineantibody production (e.g., enzyme-linked immunoassays), skin testsensitivities (e.g., subcutaneous injection of an immunogen into avaccinated animal to assess weal formation, induration and erythema),cytokine production (e.g., immunoassays using cytokine-specificantibodies or bio-assays using cytokine-dependent cell lines),antigen-specific proliferation (e.g., ³ H-thymidine incorporation),cytotoxic T cell activity (e.g., measure release of ⁵¹ Cr from targetcells), reduction of tumor burden (e.g., measure size of a tumor) and/orreduction of infectious agent burden (e.g., obtaining, for example,viral titers, bacterial colony counts or parasite counts).

An effective dose refers to a dose capable of immunizing an animalagainst an immunogen. Effective doses can vary depending upon, forexample, the adjuvant used, the immunogen being administered, and thesize and type of the recipient animal. Effective doses to treat ananimal to an immunogen include doses administered over time that arecapable of preventing or alleviating a disease in an animal to, forexample, a pathogen or allergen. For example, a first treatment dose cancomprise an amount of an adjuvant composition of the present inventionthat causes a minimal hypersensitive response when administered to ahypersensitive animal. A second treatment dose can comprise a greateramount of the same adjuvant composition than the first dose. Effectivetreatment doses can comprise increasing concentrations of the adjuvantcomposition necessary to treat an animal such that the animal does notexhibit signs of a disease.

In accordance with the present invention, a suitable single dose is adose that is capable of vaccinating an animal against a foreign agentwhen administered one or more times over a suitable time period. Forexample, a preferred single dose of an adjuvant composition of thepresent invention is from about 100 μg to about 1 mg of the adjuvantcomposition per kilogram body weight of the animal. Further treatmentswith the adjuvant composition can be administered from about 1 week toabout 1 year after the original administration. Further treatments withthe adjuvant composition preferably are administered when the animal isno longer protected from an immunogen to which the animal has beentreated. Particular administration doses and schedules can be developedby one of skill in the art based upon the parameters discussed above.

The number of doses administered to an animal is dependent upon theimmunogen and the response of an individual patient to the adjuvantcomposition. For example, treatment of one strain of virus may requiremore doses than treatment of a more immunogenic strain of virus. Thus,it is within the scope of the present invention that a suitable numberof doses includes any number required to treat an animal. A preferrednumber of doses of an adjuvant composition comprising asuperantigen-encoding recombinant molecule, and/or a cytokine-encodingrecombinant molecule and/or a chemokine-encoding recombinant molecule isfrom about 2 to about 20 administrations, preferably from about 3 toabout 10 administrations, and even more preferably from about 3 to about5 administrations per patient per year. Preferably, such administrationsare given once every 2 weeks until, for example, antibody productionagainst an immunogen increases or decreases, cell mediated immunityincreases, and/or a clinical response is observed when an adjuvantcomposition is administered as a therapeutic remedy.

A preferred single dose of the superantigen-encoding recombinantmolecule is an amount that, when transfected into a muscle cells, skintissue, lung cells or other suitable cellular sites, leads to theproduction of from about 10 femtograms (fg) to about 0.01 μg, preferablyfrom about 100 fg to about 1 picogram (pg), and more preferably fromabout 1 pg to about 5 pg of superantigen per transfected cell. Apreferred single dose of a cytokine-encoding recombinant molecule is anamount that when transfected into a target cell population leads to theproduction of from about 10 pg to about 0.01 μg, preferably from about100 fg to about 2 pg, and more preferably about 1 pg of cytokine pertransfected. A preferred single dose of a chemokine-encoding recombinantmolecule is an amount that when transfected into a target cellpopulation leads to the production of from about 1 pg to about 0.01 μg,preferably from about 0.1 pg to about 10 pg, and more preferably about 1pg of chemokine per transfected.

In one embodiment, an adjuvant composition of the present inventioncomprises up to about 50% of an immunogen-encoding recombinant moleculeand up to about 50% of a superantigen-encoding recombinant molecule.Preferably, an adjuvant composition of the present invention comprisesno more than about 1.5 mg of immunogen-encoding recombinant molecule andno more than about 1.5 mg of superantigen-encoding recombinant molecule,more preferably no more than about 1 mg of immunogen-encodingrecombinant molecule and no more than about 1 mg ofsuperantigen-encoding recombinant molecule, and even more preferably nomore than about 0.5 mg of immunogen-encoding recombinant molecule and nomore than about 0.5 mg of superantigen-encoding recombinant molecule peranimal.

In another embodiment, an adjuvant composition of the present inventioncomprises an immunogen-encoding recombinant molecule in an amount up toabout 66% by weight of the composition and a superantigen-encodingrecombinant molecule in an amount up to about 33% by weight of thecomposition. Preferably, an adjuvant composition of the presentinvention comprises no more than about 2000 μg of immunogen-encodingrecombinant molecule and no more than about 1000 μg ofsuperantigen-encoding recombinant molecule, more preferably no more thanabout 1400 μg of immunogen-encoding recombinant molecule and no morethan about 660 μg of superantigen-encoding recombinant molecule, andeven more preferably no more than about 670 μg of immunogen-encodingrecombinant molecule and no more than about 330 μg ofsuperantigen-encoding recombinant molecule per animal.

In another embodiment, an adjuvant composition of the present inventioncomprises an immunogen-encoding recombinant molecule in an amount up toabout 50% of the composition; a superantigen-encoding recombinantmolecule in an amount up to about 25% of the composition; and acytokine-encoding recombinant molecule or chemokine-encoding recombinantmolecule or mixtures thereof, in an amount up to about 25% of thecomposition. According to the present embodiment, a cytokine-encodingrecombinant molecule or a chemokine-encoding recombinant molecule can beused alone or in combination with each other. When used in combination,the ratio of cytokine-encoding recombinant molecule tochemokine-encoding recombinant molecule can be varied according to need.The ratio can be determined based upon the effectiveness of the adjuvantcomposition at vaccinating an animal against a foreign agent using themethods and parameters disclosed herein.

In one embodiment, an adjuvant composition of the present inventioncomprises: no more than about 2000 μg of immunogen-encoding recombinantmolecule, no more than about 500 μg of superantigen-encoding recombinantmolecule, and no more than about 500 μg of cytokine-encoding recombinantmolecule or no more than about 500 μg of chemokine-encoding recombinantmolecule; more preferably no more than about 1400 μg ofimmunogen-encoding recombinant molecule, no more than about 300 μg ofsuperantigen-encoding recombinant molecule, and no more than about 300μg of cytokine-encoding recombinant molecule or no more than about 300μg of chemokine-encoding recombinant molecule; and even more preferablyno more than about 660 μg of immunogen-encoding recombinant molecule, nomore than about 160 μg of superantigen-encoding recombinant molecule,and no more than about 160 μg of cytokine-encoding recombinant moleculeor no more than about 160 μg of chemokine-encoding recombinant moleculeper animal.

In another embodiment, an adjuvant composition of the present inventioncomprises: no more than about 2000 μg of immunogen-encoding recombinantmolecule, no more than about 500 μg of superantigen-encoding recombinantmolecule, and no more than about 250 μg of cytokine-encoding recombinantmolecule and no more than about 250 μg of chemokine-encoding recombinantmolecule; more preferably no more than about 1000 μg ofimmunogen-encoding recombinant molecule, no more than about 250 μg ofsuperantigen-encoding recombinant molecule, and no more than about 125μg of cytokine-encoding recombinant molecule and no more than about 125μg of chemokine-encoding recombinant molecule; and even more preferablyno more than about 660 μg of immunogen-encoding recombinant molecule, nomore than about 160 μg of superantigen-encoding recombinant molecule,and no more than about 80 μg of cytokine-encoding recombinant moleculeand no more than about 80 μg of chemokine-encoding recombinant moleculeper animal.

Adjuvant compositions are preferably delivered by intramuscularadministration in the form of "naked" DNA molecules, such as disclosedherein. Preferably, an adjuvant composition of the present invention isdelivered by intramuscular, intravenous, intraperitoneal and/orintraarterial injection, and/or injection directly into specificcellular locations (e.g., into a tumor). Preferred sites ofintramuscular injections include caudal thigh muscle, back muscle andinto the buttocks of a human.

Preferably, an adjuvant composition of the present invention comprises asuitable pharmaceutically acceptable carrier for delivering thecomposition intramuscularly. A preferred carrier for use with anadjuvant includes phosphate buffered saline, water, Ringer's solution,dextrose solution, Hank's balanced salt solution and normal saline. Amore preferred carrier includes phosphate buffered saline and normalsaline, with phosphate buffered saline being even more preferred.

Preferably, an adjuvant composition of the present invention comprises amixture including a superantigen encoding molecule including anSEA-encoding recombinant molecule, an SEB-encoding recombinant moleculeor mixtures thereof, and an immunogen-encoding recombinant molecule ofthe present invention; a superantigen encoding molecule including anSEA-encoding recombinant molecule, an SEB-encoding recombinant moleculeor mixtures thereof, a cytokine encoding molecule including aGM-CSF-encoding recombinant molecule and an immunogen-encodingrecombinant molecule of the present invention; or a superantigenencoding molecule including an SEA-encoding recombinant molecule, anSEB-encoding recombinant molecule or mixtures thereof, a chemokineencoding molecule including a MIP1α, MIP1β, IL-8 or RANTES recombinantmolecule and an immunogen-encoding recombinant molecule of the presentinvention.

In a preferred embodiment, an adjuvant of the present invention includesthe following recombinant molecules contained in phosphate bufferedsaline: (1) PCR₃ -SEA, PCR₃ -SEA.S, PCR₃ -SEB, PCR₃ -SEB.S, PCR₃ -TSSTand mixtures thereof; (2) mixtures of up to about 50% PCR₃ -SEA, PCR₃-SEA.S, PCR₃ -SEB, PCR₃ -SEB.S and/or PCR₃ -TSST, and up to about 50%PCR₃ -GM₃ ; (3) mixtures of up to about 50% PCR₃ -SEA, PCR₃ -SEA.S, PCR₃-SEB, PCR₃ -SEB.S and/or PCR₃ -TSST, and up to about 50% PCR₃ -MIP1α;(4) mixtures of up to about 50% PCR₃ -SEA, PCR₃ -SEA.S, PCR₃ -SEB, PCR₃-SEB.S and/or PCR₃ -TSST, and up to about 50% PCR₃ -MIP1β; (5) mixturesof up to about 50% PCR₃ -SEA, PCR₃ -SEA.S, PCR₃ -SEB, PCR₃ -SEB.S and/orPCR₃ -TSST, and up to about 50% PCR₃ -RANTES; (6) mixtures of up toabout 50% PCR₃ -SEA, PCR₃ -SEA.S, PCR₃ -SEB, PCR₃ -SEB.S and/or PCR₃-TSST, up to about 25% PCR₃ -GM₃, and up to about 25% PCR₃ -MIP1α, PCR₃-MIP1β and/or PCR₃ -RANTES.

According to the present invention, a preferred embodiment of anadjuvant composition of the present invention includes: (1) animmunogen-encoding recombinant molecule the present invention in anamount up to about 50% of the composition and a preferred embodiment ofan adjuvant of the present invention in an amount up to about 50% of thecomposition; or (2) an immunogen-encoding recombinant molecule in anamount up to about 66% of the composition and a preferred embodiment ofan adjuvant of the present invention in an amount up to about 33% of thecomposition, in phosphate buffered saline.

The following examples are provided for the purposes of illustration andare not intended to limit the scope of the present invention.

EXAMPLES Example 1

This example describes the production of recombinant molecules encodingsuperantigens and cytokines.

Full-length cDNA encoding Staphylococcal enterotoxin B (SEB; SEQ IDNO:1) and Staphylococcal enterotoxin A (SEA; SEQ ID NO:3) were producedby polymerase chain reaction (PCR) amplification using templatesobtained from Dr. John Kappler (National Jewish Center for Immunologyand Respiratory Disease, Denver, Colo.). A truncated form of SEB lackingthe leader sequence, which spans base pairs 46 to 773 (referred toherein as SEB.S), was prepared by PCR amplification using the primers 5'GGGAATTCCATGGAGAGTCAACCAG 3' (SEQ ID NO:7) and 3'GCGGATCCTCACTTTTTCTTTGT 5' (SEQ ID NO:8). A truncated form of SEAlacking the signal sequence, which spans base pairs 46 to 751 (referredto herein as SEA.S), was prepared by PCR amplification using the primers5' GGGAATTCCATGGAGAGTCAACCAG 3' (SEQ ID NO:9) and 5'GCAAGCTTAACTTGTATATAAATAG 3'(SEQ ID NO:10). Full-length cDNA encodingToxic Shock Syndrome Toxin (TSST; SEQ ID NO:5) was produced by PCRamplification using a template obtained from Dr. Brian Kotzin (NationalJewish Center for Immunology and Respiratory Disease, Denver, Colo.),using the primers:

5'CGGGGTACCCCGAAGGAGGAAAAAAAAATGTCTACAAACGATAATATAAAG3' (SEQ ID NO:11);and

3' TGCTCTAGAGCATTAATTAATTTCTGCTTCTATAGTTTTTAT 5' (SEQ ID NO:12).

Each cDNA clone was ligated into the eukaryotic expression vector PCR₃(In vitrogen, San Diego, Calif.) using standard cloning methods. Thefull-length SEB cDNA cloned into PCR₃ is referred to herein as PCR₃-SEB; the full-length SEA cDNA cloned into PCR₃ is referred to herein asPCR₃ -SEA; the full-length TSST cDNA cloned into PCR₃ is referred toherein as PCR₃ -TSST; the truncated SEB cDNA cloned into PCR₃ isreferred to herein as PCR₃ -SEB.S; and the truncated SEA cDNA clonedinto PCR₃ is referred to herein as PCR₃ -SEA.S.

A cDNA for canine GM-CSF was produced by PCR amplification of total RNAextracted from Concavalin A-stimulated normal canine peripheral bloodmononuclear cells (PBMC) using canine GM-CSF primers designed based onthe published canine GM-CSF cDNA (Nash, ibid.). The total RNA wasreverse transcribed using the reverse transcriptase enzyme and oligoTprimers. The canine GM-CSF cDNA was then amplified using PCR andspecific 5' and 3' primers. The PCR product was cloned into the PCR₃vector, the resulting recombinant molecule is referred to herein as PCR₃-GM₃.

Example 2

This example describes the expression of DNA encoding superantigens inmammalian CHO cells following transfection.

Isolated PCR₃ -SEB.S, PCR₃ -SEA.S and PCR₃ -TSST were transformed intoE. coli cells and ampicillin-resistant bacterial colonies were screenedfor the presence of the plasmid. Selected colonies were then cultured inlarge scale culture (liter volume). Plasmid DNA was isolated usingstandard methods. A typical plasmid yield was 20 mg plasmid DNA from oneliter of bacteria-containing culture medium. Plasmid DNA was transfectedinto Chinese hamster ovary cells (CHO) by lipofection (Lipofectamine,Gibco-BRL, Gaithersburg, Md.) using methods provided by themanufacturer. About 2.0 μg of each plasmid DNA was separatelytransfected into about 10⁶ CHO cells.

The transfected CHO cells were cultured for 48 hours. Supernatants andcell lysates were then isolated to determine the amount of intracellularand secreted SAg protein produced by the transfected cells. Cell lysateswere prepared by detaching and sonicating the transfected cells toprepare cell lysates to measure activity. SAg protein activity in eachsample was measured by quantitating the ability of the SAg protein tostimulate lymphocyte contained in a PBMC population using the followingmethod. Supernatants and lysates to be tested were added in serialdilutions to triplicate wells of a 96-well plate containing 5×10⁵ PBMCin a total volume of 200 μl per well. After 3 days, the wells werepulsed with ³ H thymidine and incubated for 18 hours. The radioactivityincorporated into the PBMC's were quantitated on a beta counter.Negative controls included CHO cells transfected with the DNA vectorwithout an inserted gene (mock) and positive controls were purifiedrecombinant SAg proteins.

The results were plotted as the mean incorporated thymidine in countsper minute and are shown in FIG. 1. The results indicate that bothsupernatants and lysates of CHO cells transfected with PCR₃ -SEB.S, PCR₃-SEA.S and PCR₃ -TSST stimulated strong proliferation of the PBMC's,compared to mock transfected cultures. The activity in supernatants insome cases exceeded that in cell lysates. Thus, DNA encoding bacterialSAg proteins are capable of being transcribed and translated inmammalian cells in biologically active form. The results also indicatethat the amounts of biologically active SAg protein are active producedby the transfected cells was sufficient to stimulate T cellproliferation.

Example 3

This example describes the expression of DNA encoding superantigens incanine melanoma cells following transfection.

A melanoma cell line was established from an oral malignant melanomaobtained by biopsy from a canine patient by isolating a portion of amelanoma tumor, digesting that portion with collagenase and plating thereleased cells in 24 well plates using Iscove Modified Dulbecco's Medium(IMDM) with 10% fetal calf serum. Melanoma cells were transfected withPCR₃ -SEB.S, PCR₃ -SEA.S and PCR₃ -TSST by lipofection as described inExample 2. The cells were then irradiated (15,000 Rads). Four samples ofeach sample of transfected melanoma cells were prepared, in whichdecreasing numbers of the transfected cells were added to normal caninePBMC (5×10⁵ /well). Each sample was prepared in triplicate in a 96 wellplate. After 3 days, proliferation was quantitated as described inExample 2. Non-transfected melanoma cells were used as negative controlsamples. The results were plotted as the mean incorporated thymidine incounts per minute and are shown in FIG. 2. The results indicate thatCanine PBMC proliferated when cultured with canine melanoma cellstransfected with PCR₃ -SEB.S, PCR₃ -SEA.S and PCR₃ -TSST, exhibiting adose-dependent increase in proliferation as increasing numbers ofirradiated tumor cells were used. Thus, melanoma tumor cells can betransfected and can express biologically active SAg protein. The resultsalso show that the transfected melanoma cells continue to releasebiologically active SAg protein after irradiation, indicating thattransfected tumor cells would also be useful as an autologous tumorvaccine as described in detail in the present application.

Example 4

This example describes the long term expression of DNA encoding SEB.Sand SEA.S in stably transfected CHO cells.

To determine whether the SAg protein activity detected in supernatantsof transfected CHO cells (described in Example 2) represented actualsecretion or simple release from dying cells, stably transfected CHOlines were prepared using either PCR₃ -SEB.S, PCR₃ -SEA.S or vector withno cDNA insert (control). About 2×10⁶ CHO cells were transfected withabout 2 μg of plasmid DNA by lipofection. The transfected cells werethen cultured in G418 (1 mg/ml) for 3 weeks to obtain stabletransfectants. The G418 selected CHO cells were seeded into 9 individualtissue culture wells, allowed to adhere for 4 hours, and then freshtissue culture media was added. Supernatants were harvestedsequentially, beginning at time zero and continuing for 36 hours.Supernatants were added to PBMC to assay for SAg protein activity, asdescribed in Example 2.

The results were plotted as the mean proliferation stimulating activitycontained in supernatants at each time point and are shown in FIGS. 3Aand 3B. The results indicate that a steady time-dependent increase inPBMC stimulatory activity was observed in supernatants from CHO cellsstably transfected with both PCR₃ -SEB.S and PCR₃ -SEA.S. Thus,transfection of mammalian cells with PCR₃ -SEB.S, PCR₃ -SEA.S results inlong term expression of biologically active SAg protein. The dataindicates that transfected mammalian cells can serve as a sustainedsource of SAg protein production.

Example 5

This example describes that transfection of PCR₃ -SEA.S DNA in melanomacells results in the expression of biologically active SEA.S protein.

Superantigens are capable of stimulating the proliferation of T cellsbearing certain Vβ domains in their T cell receptor (TCR). SEA is knownto stimulate T cells having a Vβ3+ TCR in mice. SEB does not stimulateVβ3+ T cells. Therefore, an experiment was performed to assess theability of SEA.S protein expressed by melanoma cells transfected withPCR₃ -SEA.S DNA to stimulate the proliferation of a T cell clone (AD10)expressing the Vβ3+TCR.

B16 melanoma cells were transfected with PCR₃ -SEA.S DNA, PCR₃ -SEB.S orPCR₃ vector DNA with no insert (mock). The cells were then irradiated(18,000 Rads) and plated in triplicate in a 96 well plate at aconcentration of about 1×10⁴ per well. About 1×10⁵ AD10 cells were addedto each well. Next, irradiated syngeneic spleen cells were added to eachwell as a source of antigen presenting cells for the superantigen and Tcell interaction. Negative controls included mock transfected cells;positive controls included recombinant SEA (10 ng/ml). The cells wereincubated for 48 hours. ³ H thymidine was then added to each well andthe proliferative response quantitated.

The results were plotted as the mean incorporated thymidine in countsper minute and are shown in FIG. 4. The AD10 cells proliferated stronglyin response to SEA.S protein produced by the PCR₃ -SEA.S DNA transfectedinto the B16 cells, with the proliferative response nearly equal to thatof the recombinant protein. Thus, the T cell response generated bytransfection of melanoma cells with PCR₃ -SEA.S DNA is specific for thecorrect TCR. Cells transfected with PCR₃ -SEB.S DNA did not stimulateproliferation of AD10 cells, consistent with the predicted TCRspecificity of SEA and SEB.

Example 6

This example describes the expression of PCR₃ -GM DNA in CHO cells.

PCR₃ -GM DNA was produced, isolated and transfected into CHO cells usingthe methods described in Examples 1 and 2. Expression of GM-CSF proteinin the CHO cells was measured by the following method. Supernatants wereisolated from the cultures of the transfected cells and non-transfectedCHO cells. The supernatants were added to cultures of monocyte cells(obtained from normal canine PBMC) and the ability of the supernatantsto support the growth and survival of monocytes was determined. After 4days in culture with test or control CHO supernatants, monocyte survivalwas quantitated by addition of methyltetrazolium dye (MTT) that isreduced in viable cells. Absorbance of light at 570 nm (measured usingan ELISA reader) is representative of cell survival.

The results are shown in FIG. 5 and indicate that the supernatants fromCHO transfected with PCR₃ -GM DNA stimulated the survival of caninemonocytes in culture compared with results obtained using the controlsupernatants. The level of activity was comparable to that of 1×10⁵units of canine recombinant GM-CSF. Thus, the GM-CSF protein produced byCHO cells transfected with PCR₃ -GM DNA is biologically active.

Example 7

This example demonstrates that the vaccination of mice with autologoustumor cells transfected with PCR₃ -SEA.S DNA or PCR₃ -SEB.S DNA inducestrong cytotoxic T cell (CTL) activity.

The following experiment studies the ability of non-immunogenic murinemelanoma cells (B16 melanoma cells, F10 clone) expressing either PCR₃-SEA.S DNA or PCR₃ -SEB.S to induce CTL responses in mice. B16 cells areknown to be non-immunogenic when injected into C57B16/J mice. The levelof CTL responses that can be induced has been shown to correlate withthe ability of the immunized animal to reject tumors.

B16 cells were transfected with either PCR₃ -SEA.S DNA, PCR₃ -SEB.S orPCR₃ DNA lacking insert (mock) using the method described in Example 2.The cells were then irradiated at 12,000 Rads. About 10⁶ irradiatedcells were then injected subcutaneously into C57B16/J mice. Three weekslater, the mice were sacrificed and their spleen mononuclear cellsharvested. Mononuclear cells isolated from the spleen cells were thenrestimulated in vitro with irradiated, non-transfected wild type B16cells for 6 days in media with interleukin-2 (IL-2). The spleen cellswere then added in decreasing numbers to about 5×10³ of ⁵¹ Cr-labeledwild type (non-transfected) B16 cells in a standard chromium releaseassay for CTL activity. After 4 hours, the supernatants were harvestedand the percentage of specific lysis of the target B16 melanoma cellswas quantitated.

The results are shown in FIGS. 6A and 6B and indicate that injection ofanimals with irradiated transfected melanoma cells induce greater CTLactivity than injection with non-transfected cells. This result isconsistent with the non-immunogenic nature of B16 cells. Thus, DNAencoding bacterial SAg proteins expressed in transfected tumor cells arecapable of eliciting strong CTL-mediated immunity against thenon-transfected parental cell. These results suggest that autologoustumor cells transfected with DNA encoding a superantigen constitute aneffective tumor vaccine for treatment or prevention of metastaticdisease.

Example 8

This example demonstrates that tumor cells transfected with PCR₃ -SEB.SDNA are capable of inducing cytotoxic activity in adjacent T cells.

T cells were prepared from a mouse immunized with non-transfected B16cells using the methods described in Example 7. These isolated cellsexhibited minimal CTL activity towards non-transfected B16 target cells.B16 cells were transfected with PCR₃ -SEB.S using the methods generallydescribed in Example 2. Induction of CTL activity by the transfected B16target cells was assessed in a standard 4 hour chromium release assay asused in Example 7.

The results are shown in FIG. 7 and indicates that B16 cells transfectedwith PCR₃ -SEB.S produced protein that rapidly induced a four-foldincrease in CTL activity in T cells that were relatively unresponsive tonon-transfected target B16 cells. Thus, the SEB produced in the vicinityof the isolated T cells by the B16 cells is capable of stimulating suchT cells. The data indicates that tumor cells transfected in vivo withPCR₃ -SEB.S can produce biologically active SEB.S that is capable ofrapidly activating T lymphocytes in their vicinity and thereby inducingcytotoxic activity against themselves or neighboring tumor cells.

Example 9

This example describes the treatment of canine melanoma with DNAencoding superantigen or GM-CSF.

A. Criteria for entry and trial design

Animals selected for entry into the present study were client ownedanimals with spontaneous oral malignant melanoma, a highly malignantneoplasm of dogs for which there is no alternative effective treatment.Prior to entry, the owners were required to sign informed consent. Thestudy consisted of an initial 12 week trial response phase with 6injections given once every 2 weeks, followed by long term once monthlymaintenance therapy for those animals that responded during the initial12 week induction phase. Potential toxicity was assessed by (1) bodytemperature measured daily for 7 days after injection; (2) physicalexamination of the injection site; (3) owner's assessment of their pet'sattitude and appetite; (4) complete blood counts and biochemistrymeasurements once monthly. Treatment responses were assessed by: (1)physical measurement of tumor dimensions; (2) tumor photography; (3)thoracic radiographs for metastasis evaluation.

B. Superantigen+GM-CSF Treatment protocol

DNA samples complexed with liposomes were prepared as follows. PCR₃-SEB.S and PCR₃ -GM plasmid DNA prepared from bacterial cultures by thealkaline lysis method, then purified by CsCl banding, were resuspendedat a 1.0 mg/ml concentration in sterile PBS. Liposomes were prepared bymixing equimolar amounts of N-1-(33-dioleyloxy)propyl!-N,N,N-triethylammonium (DOTMA; obtained fromSyntex, Corp., Palo Alto, Calif.) and dioleoyl phosphatidylethanolamine(DOPE; obtained from Avanti Polar Lipids, Birmingham, Ala.). The lipidswere dried in a desiccator and reconstituted at a concentration of 1.0mg/ml in sterile phosphate buffered saline (PBS), pH 7.0. Thereconstituted lipids were sonicated for 5 minutes to produce liposomeshaving an average size of about 200 nm to about 400 nm.

Thirty minutes prior to injection into the patients, the PCR₃ -SEB.S andPCR₃ -GM DNA was mixed with the liposomes at a ratio of 1.0 μg DNA to 4nmol liposome, in 1.0 ml sterile PBS. The solution was allowed tocomplex at room temperature. Two doses of DNA were administered,depending on tumor volume. For tumors less than 3 centimeters (cm) indiameter, 400 μg total DNA (200 μg each of PCR₃ -SEB.S and PCR₃ -GM DNA)were injected into each tumor. For tumors larger than 3 cm diameter, atotal of 800 μg DNA (400 μg each of PCR₃ -SEB.S and PCR₃ -GM DNA) wereinjected into each tumor.

For each treatment, the DNA:liposome mixture was injected into the tumorsite with a 3 ml syringe and 25 gauge needle. For larger tumors, most ofthe injection was delivered into tissues at the periphery of the tumorbase. For some smaller tumors, injections were also injected directlyinto tumor tissue. Lymph node tissue having evidence of tumor metastasiswas also injected. Injections were performed once every 2 weeks for thefirst 12 weeks, then continued twice monthly for those animals in whichan initial treatment response occurred, until complete tumor regressionoccurred. At that time, the frequency of injections decreased to oncemonthly. The toxicity of the treatment was evaluated based on theparameters outlined above in section A. The results are shown below inTable 1.

                                      TABLE 1    __________________________________________________________________________    Patient Log for SEB.S and PCR.sub.3 -GM DNA Treatment of Canine Melanoma    Patient         Stage            TN    Tumor Size                         Start Date                              Response                                    Comments    __________________________________________________________________________    Zomax         I  T1bNOMO                  1.5 cm diam                         5/16/94                              CR 51 wks                                    SEB.S + GM-CSF    Shadow         III            T2bN1bMO                  3 cm diam                         5/23/94                              CR 50 wks                                    SEB.S + GM-CSF    NG   I  T1NOMO                  1.2 cm diam                         9/12/94                              CR 34 wks                                    SEB.S + GM-CSF    Maggie         II T2aNOMO                  2 cm diam                         8/24/94                              PR 33 wks                                    SEB.S + GM-CSF    K.C. III            T3aNOMO                  >4 cm diam                         10/13/94                              SD 12 wk                                    SEB.S + GM-CSF    Belvedere         III            T2N1bMO                  4 cm diam                         10/13/94                              CR 30 wks                                    SEB.S + GM-CSF    Nicholas         III            T3bNOMO                  >4 cm diam                         2/15/95                              SD 12 wks                                    SEB.S + GM-CSF    Heidi         III            TON1bMO                  LN: 2 cm diam                         2/27/95                              PR 10 wks                                    SEB.S + GM-CSF    Bear III            TON1bMO                  LN: 2.5 cm                         4/11/95                              SD 4 wks                                    SEB.S + GM-CSF    __________________________________________________________________________     Key to terminology in patient data sheets:     Stage: I represents the smallest and III the largest size, with metastase     TNM: World Health Organization staging system     SD = stable disease (no tumor growth)     PR = partial remission (>50% decrease in tumor size)     CR = tumor completely regressed     PD = progressive disease, no response to treatment     MCT = mast cell tumor     Mammary CA = mammary gland adenocarcinoma (malignant breast cancer)     Thyroid CA = thyroid adenocarcinoma     SCC = squamous cell carcinoma

The results shown in Table 1 indicate that a treatment response wasobserved in 6 of 9 dogs treated for the 12 week trial period. Thisincluded 4 complete remissions (no residual tumor) and 2 partialremissions (greater than 50% reduction in tumor size). Tumors in theremaining two dogs did not regress, but also did not progress in sizeduring the 12 week trial. On average, a tumor response required 6 to 10weeks to become apparent. The injections did not cause any inflammationor necrosis at injections sites. Toxicity, either local or systemic, wasnot observed in any of the 10 patents treated in this study. Theseresults provide evidence of the efficacy of direct DNA injection usingDNA encoding superantigen (SEB) and cytokine (GM-CSF) for treatment ofspontaneous malignant melanoma in an outbred species.

Canine melanoma is a highly malignant, rapidly growing tumor of dogs,and provides a useful model for the study of treatments for humanmelanoma. Without treatment, the 50% survival time for animals withstage III disease (5 of the patients in this study) is about 3 monthsand all animals will be dead by 5 months due to pulmonary metastases.The observation of several long term survivors shown in Table 1 (othershave not been treated long enough to evaluate) suggests that thecombined DNA immunotherapy approach also has a systemic effect onpreventing metastatic diseases.

Another major advantage of this approach is the apparent completeabsence of toxicity in the dogs. Since dogs respond to SAg proteinsimilar to humans, it is also likely that toxicity in humans would alsobe minimal. The delivery of DNA encoding superantigens into tumor cellsby transfection and subsequent local expression is sufficient to inducea strong immune response without inducing toxicity. Thus this geneticapproach to tumor immunotherapy offers advantages over conventionalchemotherapy and radiation therapy in terms of reducing patientmorbidity. In addition, delivering the SAg protein by DNA transfectionalso avoids the potential toxicity associated with systemicadministration.

C. Single Gene Treatment Protocol

To evaluate the effectiveness of injecting DNA encoding either asuperantigen or a cytokine, relative to combined genetic therapy(SAg-encoding DNA and cytokine-encoding DNA), 2 groups of dogs weretreated with either PCR₃ -SEB.S DNA alone (3 dogs) or PCR₃ -GM DNA alone(3 dogs; 2 entered, one evaluatable). Similar criteria for entry andtrial design described above in Section A of this example was applied.Although not formally randomized, after the first 10 dogs were treatedwith the 2 gene combination, the next 3 enrollees were assigned the PCR₃-SEB.S DNA alone group and the next 3 to the PCR₃ -GM DNA alone group. Asimilar treatment protocol as described above in section B was appliedin this study. Briefly, the DNA was complexed with liposomes andinjected once every 2 weeks for the first 12 weeks, then continued twicemonthly for those animals in which an initial treatment responseoccurred, until complete tumor regression occurred. The toxicity of thetreatment was evaluated based on the parameters outlined above insection A. The results are shown below in Table 2.

                                      TABLE 2    __________________________________________________________________________    Patient Log for SEB.S or PCR.sub.3 -GM DNA alone Treatment of Canine    Melanoma    Patient         Stage            TN    Tumor Size                         Start Date                              Response                                    Comments    __________________________________________________________________________    Jessie         II T2bNOMO                  2 cm diam                         1/11/95                              PD 17 wks                                    SEB.S alone    Mr. T         III            TON1bMO                  LN: 2 cm diam                          2/1/95                              PD 14 wks                                    SEB.S alone    Duffy         II T2aNOMO                  2 cm diam                          2/3/95                              PD 12 wks                                    SEB.S alone    Scooter         I  T2aNOMO                  2 cm diam                         3/24/95                              PD 7 wks                                    GM-CSF alone    __________________________________________________________________________

The results indicated that a tumor response did not occur in any dogreceiving PCR₃ -SEB.S DNA alone and tumors grew progressively. Inaddition, one dog (Scooter) treated with PCR₃ -GM DNA alone alsoexhibited progressive growth. These data indicate that treatment withPCR₃ -SEB.S DNA alone or PCR₃ -GM DNA alone does not induce tumorregression. The data indicate that the marked anti-tumor efficacy ofdirect DNA injection results from the combined expression of PCR₃ -SEB.SDNA and PCR₃ -GM DNA in a tumor and adjacent tissues.

Example 10

This example describes the treatment of various tumor types withsuperantigen or GM-CSF encoding DNA.

The efficacy and lack of toxicity of PCR₃ -SEB.S DNA and PCR₃ -GM DNAwas determined for the treatment of dogs with malignancies havingsimilar biological and histological characteristics as human cancers.Dogs with five different cancers (advanced mammary carcinoma, mast celltumor, thyroid carcinoma, non-oral melanoma, and squamous cellcarcinoma) were treated in this study. Animals selected for entry intothe present study included dogs with spontaneous malignancies that hadreceived alternative treatments (e.g., chemotherapy and/or surgery) andeither, had not responded, or had relapsed.

Therapeutic samples were prepared and injected intratumorally with PCR₃-SEB.S DNA and PCR₃ -GM DNA as described above in Example 2. The dogswere treated initially once every 2 weeks for 12 weeks, then continuedtwice monthly for those animals in which an initial treatment responseoccurred. The toxicity of the treatment was evaluated based on theparameters outlined above in Example 9, section A. The results are shownbelow in Table 3.

                                      TABLE 3    __________________________________________________________________________    Patient Log for SEB.S and PCR.sub.3 -GM DNA Treatment of Various    Carcinomas                              Start    Patient        Tumor Type               Stage                  TN    Tumor Size                              Date Response                                         Comments    __________________________________________________________________________    Emma        Mammary CA               III                  T4N1bNMO                        1.8 cm diam                              8/11/94                                   PR 22 wks                                         SEB.S + GM-CSF    Baby        Mammary CA               II T1aN1bMO                        2.6 cm diam                              9/12/94                                   PR 8 wks                                         SEB.S + GM-CSF    Christa        MCT    IIIa                  NA    >2 cm diam                              7/27/94                                   SD 39 wks                                         SEB.S + GM-CSF    Jack        MCT    IIIa                  NA    >3 cm diam                              3/28/95                                   PD 4 wks                                         SEB.S + GM-CSF    Britt        Thyroid CA               III                  T3bNoMo                        >7 cm diam                              10/14/94                                   SD 16 wk                                         SEB.S + GM-CSF    Duncan        Melanoma Toe               NA*                  T2N1MO                        >4 cm diam                              8/11/94                                   SD 20 wks                                         SEB.S + GM-CSF    Billy        Melanoma Toe               NA*                  TON1bMO                        LN 3.5 cm                              1/10/95                                   CR 17 wks                                         SEB.S + GM-CSF    Scotche        SCC Tonsil               NA T3NOMO                        4 cm diam                              3/27/95                                   SD    SEB.S + GM-CSF    __________________________________________________________________________     *Metastases     NA Not Applicable     CA Carcinoma     MCT Mast Cell Tumor     SCC Squamous Cell Carcinoma

In this study, toxicity was not observed in any of the animals. Tumorresponses (partial remission of the primary tumors) were observed in theanimals with mammary carcinoma and neither animal developed additionalmetastatic disease during the course of the study. Treatment of one dog(Billy) with a large, metastatic (lymph node metastases), non-oralmelanoma resulted in complete remission of the cancer. Treatment of theother dog (Duncan) with a large, metastatic (lymph node metastases),non-oral melanoma resulted in prolonged stabilization of the disease.The dog with thyroid cancer (Britt) also experienced prolongedstabilization of the disease with once monthly injections. The responserate for the dogs with mast cell tumors was low. The effectiveness ofthe treatment on the squamous cell carcinoma is in early stages ofevaluation. Taken together, the results indicate that PCR₃ -SEB.S DNAand PCR₃ -GM DNA can effectively treat multiple tumor types, in additionto the melanomas reported above in Example 9.

Example 11

This example describes the injection of PCR₃ -SEA.S DNA into musclecells which induces potent, long-lasting T cell deletion.

Four groups of mice B10.BR (2-3 mice per group) were prepared asfollows. Group (1) consisted of untreated mice (control mice). Group (2)consisted of mice injected intraperitoneally with 100 ng of recombinantSEA (rSEA) protein. Group (3) consisted of mice injected intramuscularlywith 100 μg of PCR₃ -SEA.S DNA (50 μg per leg, total of 100 μg/mouse).Group (4) consisted of mice injected intramuscularly with 100 μg PCR₃(no insert; mock) DNA (50 μg per leg, total of 100 μg/mouse). The DNAsamples were prepared by diluting 100 μl of a solution containing 100 μgof DNA 50:50 (v:v) in sterile PBS prior to injection. The rSEA proteinwas purified from cultures of E. coli cells transformed with therecombinant molecule PKK223 (obtained from Dr. John Kappler) encodingthe truncated SEA.S protein lacking a leader sequence.

Beginning 72 hours after injection, mice were tail bled and PBMCprepared for fluorescence activated cell sorter (FACS) analysis. Cellswere double labeled with the monoclonal antibodies FITC conjugated-GK1.5antibody, biotinylated-KJ25 antibody and biotinylated-F23.1, to analyzefor expression of CD4, TCR Vβ3 and TCR Vβ8 expression, respectively. Thelabelled cells were analyzed on an EPICS-C flow cytometer.

The percentage of cells isolated from the experimental mice expressingCD4 that also expressed either Vβ8 or Vβ3 was calculated and compared topercentages expressed by cells isolated from control mice. The meanpercentage of CD4+ and Vβ3+ T cells in PBMC was plotted against timeafter injection.

The results are shown in FIG. 8 and indicate that the percentage ofCD4+, Vβ3+ T cells declined rapidly in PBMC of mice that receivedintramuscular injections with PCR₃ -SEA.S DNA, but not in mice mockinjected with mock DNA. The percentages of Vβ8+ cells was not affected.This result is predicted since SEA protein does not bind mouse Vβ8+ Tcells. The decline of the percentage of CD4+, Vβ3+ T cells occurred asrapidly as in mice injected with the recombinant SEA protein (rSEA). Thedeletion, however, observed over the next 2 months in mice injected withPCR₃ -SEA.S DNA was longer lasting and was more pronounced than thedeletion induced by injection of SEA.S protein. In addition, injectionof as little as 2 μg PCR₃ -SEA.S DNA also induced deletion of Vβ3+ Tcells. Thus, intramuscular injection of DNA encoding superantigensrepresents a potent and non-toxic approach to the deletion orsuppression of potentially harmful (e.g., autoreactive T cells) T cells.

Example 12

This example describes the production of immunogen and chemokineencoding recombinant molecules.

Recombinant molecules encoding ovalbumin (OVA) were produced by ligatingcDNA encoding OVA into the eukaryotic expression vector PCR₃ and isreferred to herein as PCR₃ -OVA. cDNA encoding murine RANTES, murinemacrophage inflammatory protein-1 alpha (MIP-1α), and macrophageinflammatory protein-1 beta (MIP-1β) was prepared from RNA isolated fromLPS-stimulated normal murine bone marrow macrophages using methodsstandard in the art. The cDNA were ligated into the expression vectorPCR₃, and are referred to herein as PCR₃ -RANTES, PCR₃ -MIP-1α and PCR₃-MIP-1β. All plasmid DNA were purified by cesium chloride gradientcentrifugation and resuspended at 1.0 mg/ml in sterile PBS.

Example 13

This example demonstrates that the co-administration of adjuvant DNA andimmunogen DNA stimulates antibody production against the immunogenprotein.

Separate groups of 4 CB6 F1 mice per group were injected twice with thefollowing mixtures of DNA: (1) about 100 μg PCR₃ -OVA+about 100 μg PCR₃-MIP-1β; (2) about 100 μg PCR₃ -OVA+about 50 μg PCR₃ -SEB (described inExample 1)+PCR₃ -GM-CSF (described in Example 1); (3) about 100 μg PCR₃-OVA+about 100 μg PCR₃ -RANTES; (4) about 100 μg PCR₃ -OVA+about 100 μgPCR₃ -SEB; (5) about 100 μg PCR₃ -OVA+about 100 μg PCR₃ -GM-CSF; or (6)about 100 μg PCR₃ -OVA alone. Control samples were also prepared whichincluded 6 non-injected, syngeneic mice. The DNA was diluted to a finalconcentration of 0.5 mg/ml in sterile phosphate buffered saline (PBS)prior to injection. The mice were injected intramuscularly, bilaterallyin their quadriceps muscles (about 100 μg of DNA per quadricep).

About 20 days after the immunization of step B, serum was collected fromeach mouse and assayed for antibodies that specifically bind to OVAprotein using an OVA-specific enzyme linked immunoassay (ELISA) assayusing methods standard in the art. Briefly, OVA protein was bound to anELISA plate. The plates were washed and then incubated in the presenceof serum. Again the plates were washed and then incubated in thepresence of HRP-conjugated anti-mouse IgG antibody. The amount ofantibody bound to the OVA was detected on an ELISA reader and areexpressed in absorbance units.

The results of the ELISA are shown in FIG. 9 and indicate thatco-injection of DNA encoding OVA, with either DNA encoding RANTES orMIP-1β, or SEB and GM-CSF, increases the antibody response to OVA overthat observed with OVA alone, OVA plus GM-CSF, OVA plus SEB alone orcontrol samples. Thus, the expression of RANTES, MIP-1β, or SEB andGM-CSF increase the antibody response to OVA when administered as a DNAvaccine.

Example 14

This example demonstrates that the co-administration of DNA adjuvant andimmunogen DNA results in the production of interferon gamma.

Separate groups of 4 CB6 F1 mice per group were injected twice,intramuscularly (on day 1 and day 21), with the following mixtures ofDNA: (1) about 100 μg PCR₃ -OVA+about 100 μg PCR₃ -MIP-1β; (2) about 100μg PCR₃ -OVA+about 50 μg PCR₃ -SEB+PCR₃ -GM-CSF; (3) about 100 μg PCR₃-OVA+about 100 μg PCR₃ -RANTES; (4) about 100 μg PCR₃ -OVA+about 100 μgPCR₃ -SEB; (5) about 100 μg PCR₃ -OVA+about 100 μg PCR₃ -GM-CSF; or (6)about 100 μg PCR₃ -OVA alone. Control samples were also prepared asabove.

The mice were sacrificed on day 27. Spleen cells were harvested fromeach mouse and re-stimulated in vitro with irradiated OVA-transfectedcells (EG7-OVA) in quadruplicate wells. On day 4 of the re-stimulationwith irradiated EG7-OVA cells, supernatants were harvested from thecultures and assayed for interferon gamma activity using an interferongamma-specific ELISA assay. Results were expressed as units/ml ofinterferon activity, as determined by comparison with a standard curvegenerated with recombinant murine interferon-gamma.

The results are shown in FIG. 10 and indicate that RANTES or GM-CSF wereeffective compounds for inducing interferon-gamma production. Althoughless, SEB and MIP-1β also induced interferon-gamma production.Additional experiments indicated that none of the adjuvants evaluated inthis experiment induced significant quantities of IL-4 release.Together, these data indicate that the immune response induced by anadjuvant of the present invention is primarily a Th1 response, whichinduces primarily cell-mediated immunity, including macrophageactivation, enhanced T cell CTL activity, and increased MHC expression.

Example 15

This example demonstrates that the co-administration of adjuvant DNA andimmunogen DNA induce T cell proliferative responses to the immunogen.

Separate group of 4 CB6 F1 mice per group were immunized using theprotocol described in Example 14. The animals were sacrificed on day 27and harvested spleen cells re-stimulated using the method described inExample 14. After about 4 days of re-stimulation, 100 μl aliquots of thecells were harvested from each well and pulsed for 18 hours with ³H-thymidine. Thymidine incorporation was then quantitated (cpm) as ameasure of the proliferative response to OVA expressed by thetransfected EG7-OVA cell line.

The results are shown in FIG. 11 and indicate that MIP-1β, RANTES,SEB+GM-CSF, and SEB alone, when co-administered together with OVA DNA,induce a substantial increase in the proliferative response to OVA.Thus, these data provide evidence that DNA encoding chemokines and SAgsare useful for enhancing cell-mediated immune responses and thereforeare useful as DNA vaccine adjuvants.

Example 16

This example demonstrates that the co-administration of adjuvant DNAincreases CTL responses to the immunogen ovalbumin.

Mice were immunized using the protocol described in Example 14. Spleencells were harvested from the immunized mice 7 days after the lastvaccination. The cells were then re-stimulated in vitro for 6 days withirradiated EG7-OVA cells. T cells were then harvested from there-stimulated population and added in decreasing numbers to ⁵¹Cr-labeled EG7-OVA or EL-4 target cells in a standard 4 hour chromiumrelease assay for CTL activity. The percent cell lysis was determinedChromium release was then quantitated (cpm) as a measure of the percentspecific cell lysis of labeled target cells. The higher the % specificlysis, the more CTL activity exhibited by the T cells.

The results are shown in FIG. 12 and indicate that all of the adjuvantDNAs evaluated induced increased CTL activity compared to OVA alone. Theuse of RANTES, GM-CSF and SEB alone, each were effective in inducing CTLactivity. These data indicate that co-administration of chemokine DNAcan enhance CTL-mediated immunity to an intracellular immunogen, astypified by OVA expressed in a transfected cell line, indicating thatthis approach is useful for vaccines against intracellular pathogens.

Taken together, the results of Examples 12-16 indicate that all DNAadjuvants tested (GM-CSF, SEB, SEB+GM-CSF, RANTES and MIP-1β) improvedcell mediated immunity against the immunogen ovalbumin. In particular,the use of either SEB or GM-CSF alone, as well as the combination ofSEB+GM-CSF were effective at inducing cell mediated immunity.

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#r Thr Lys Lys Lys    #               255    - (2) INFORMATION FOR SEQ ID NO:3:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 751 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: protein    -     (ix) FEATURE:              (A) NAME/KEY: CDS              (B) LOCATION: 46..747    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:    #GAG AAA         54ATTT AATACGACTC ACTATAGGGA ATTCC ATG    #              Met Glu L - #ys    #                1    - AGC GAA GAA ATA AAT GAG AAA GAT CTG CGC AA - #G AAG TCC GAA TTG CAG     102    Ser Glu Glu Ile Asn Glu Lys Asp Leu Arg Ly - #s Lys Ser Glu Leu Gln    #      15    - GGA ACA GCC CTA GGC AAT CTT AAA CAA ATC TA - #T TAT TAC AAT GAA AAA     150    Gly Thr Ala Leu Gly Asn Leu Lys Gln Ile Ty - #r Tyr Tyr Asn Glu Lys    # 35    - GCG AAG ACT GAG AAT AAA GAG AGT CAC GAT CA - #A TTT CTG CAG CAT ACT     198    Ala Lys Thr Glu Asn Lys Glu Ser His Asp Gl - #n Phe Leu Gln His Thr    #                 50    - ATA TTG TTT AAA GGC TTT TTT ACT GAT CAT TC - #G TGG TAT AAC GAT TTA     246    Ile Leu Phe Lys Gly Phe Phe Thr Asp His Se - #r Trp Tyr Asn Asp Leu    #             65    - CTA GTA GAT TTT GAT TCG AAG GAC ATC GTT GA - #T AAA TAT AAA GGG AAG     294    Leu Val Asp Phe Asp Ser Lys Asp Ile Val As - #p Lys Tyr Lys Gly Lys    #         80    - AAG GTC GAC TTG TAT GGT GCT TAT TAT GGG TA - #C CAA TGT GCT GGT GGT     342    Lys Val Asp Leu Tyr Gly Ala Tyr Tyr Gly Ty - #r Gln Cys Ala Gly Gly    #     95    - ACA CCA AAC AAA ACA GCA TGC ATG TAT GGT GG - #G GTA ACC TTA CAT GAC     390    Thr Pro Asn Lys Thr Ala Cys Met Tyr Gly Gl - #y Val Thr Leu His Asp    100                 1 - #05                 1 - #10                 1 -    #15    - AAT AAT CGA TTG ACC GAA GAG AAA AAG GTC CC - #G ATC AAT TTA TGG CTA     438    Asn Asn Arg Leu Thr Glu Glu Lys Lys Val Pr - #o Ile Asn Leu Trp Leu    #               130    - GAC GGT AAA CAA AAT ACA GTA CCT CTA GAA AC - #G GTT AAA ACG AAT AAG     486    Asp Gly Lys Gln Asn Thr Val Pro Leu Glu Th - #r Val Lys Thr Asn Lys    #           145    - AAA AAT GTA ACT GTC CAA GAG CTG GAT CTT CA - #A GCG CGC CGA TAC CTA     534    Lys Asn Val Thr Val Gln Glu Leu Asp Leu Gl - #n Ala Arg Arg Tyr Leu    #       160    - CAG GAA AAA TAT AAT TTG TAC AAC TCT GAC GT - #C TTT GAT GGG AAG GTT     582    Gln Glu Lys Tyr Asn Leu Tyr Asn Ser Asp Va - #l Phe Asp Gly Lys Val    #   175    - CAG AGA GGC CTA ATC GTG TTT CAT ACT TCT AC - #A GAA CCT TCG GTT AAC     630    Gln Arg Gly Leu Ile Val Phe His Thr Ser Th - #r Glu Pro Ser Val Asn    180                 1 - #85                 1 - #90                 1 -    #95    - TAC GAT TTA TTT GGA GCT CAA GGA CAG TAT TC - #A AAT ACA CTC TTA AGA     678    Tyr Asp Leu Phe Gly Ala Gln Gly Gln Tyr Se - #r Asn Thr Leu Leu Arg    #               210    - ATA TAT CGC GAC AAC AAG ACG ATT AAC TCT GA - #A AAC ATG CAC ATT GAT     726    Ile Tyr Arg Asp Asn Lys Thr Ile Asn Ser Gl - #u Asn Met His Ile Asp    #           225    #              751 CA AGT TAAGCTT    Ile Tyr Leu Tyr Thr Ser            230    - (2) INFORMATION FOR SEQ ID NO:4:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 233 amino              (B) TYPE: amino acid              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: protein    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:    - Met Glu Lys Ser Glu Glu Ile Asn Glu Lys As - #p Leu Arg Lys Lys Ser    #                 15    - Glu Leu Gln Gly Thr Ala Leu Gly Asn Leu Ly - #s Gln Ile Tyr Tyr Tyr    #             30    - Asn Glu Lys Ala Lys Thr Glu Asn Lys Glu Se - #r His Asp Gln Phe Leu    #         45    - Gln His Thr Ile Leu Phe Lys Gly Phe Phe Th - #r Asp His Ser Trp Tyr    #     60    - Asn Asp Leu Leu Val Asp Phe Asp Ser Lys As - #p Ile Val Asp Lys Tyr    # 80    - Lys Gly Lys Lys Val Asp Leu Tyr Gly Ala Ty - #r Tyr Gly Tyr Gln Cys    #                 95    - Ala Gly Gly Thr Pro Asn Lys Thr Ala Cys Me - #t Tyr Gly Gly Val Thr    #           110    - Leu His Asp Asn Asn Arg Leu Thr Glu Glu Ly - #s Lys Val Pro Ile Asn    #       125    - Leu Trp Leu Asp Gly Lys Gln Asn Thr Val Pr - #o Leu Glu Thr Val Lys    #   140    - Thr Asn Lys Lys Asn Val Thr Val Gln Glu Le - #u Asp Leu Gln Ala Arg    145                 1 - #50                 1 - #55                 1 -    #60    - Arg Tyr Leu Gln Glu Lys Tyr Asn Leu Tyr As - #n Ser Asp Val Phe Asp    #               175    - Gly Lys Val Gln Arg Gly Leu Ile Val Phe Hi - #s Thr Ser Thr Glu Pro    #           190    - Ser Val Asn Tyr Asp Leu Phe Gly Ala Gln Gl - #y Gln Tyr Ser Asn Thr    #       205    - Leu Leu Arg Ile Tyr Arg Asp Asn Lys Thr Il - #e Asn Ser Glu Asn Met    #   220    - His Ile Asp Ile Tyr Leu Tyr Thr Ser    225                 2 - #30    - (2) INFORMATION FOR SEQ ID NO:5:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 582 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: protein    -     (ix) FEATURE:              (A) NAME/KEY: CDS              (B) LOCATION: 1..582    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:    - ATG ACA AAC GAT AAT ATA AAG GAT TTG CTA GA - #C TGG TAT AGT AGT GGG      48    Met Thr Asn Asp Asn Ile Lys Asp Leu Leu As - #p Trp Tyr Ser Ser Gly    #                 15    - TCT GAC ACT TTT ACA AAT AGT GAA GTT TTA GA - #T AAT TCC TTA GGA TCT      96    Ser Asp Thr Phe Thr Asn Ser Glu Val Leu As - #p Asn Ser Leu Gly Ser    #             30    - ATG CGT ATA AAA AAC ACA GAT GGC AGC ATC AG - #C CTT ATA ATT TTT CCG     144    Met Arg Ile Lys Asn Thr Asp Gly Ser Ile Se - #r Leu Ile Ile Phe Pro    #         45    - AGT CCT TAT TAT AGC CCT GCT TTT ACA AAA GG - #G GAA AAA GTT GAC TTA     192    Ser Pro Tyr Tyr Ser Pro Ala Phe Thr Lys Gl - #y Glu Lys Val Asp Leu    #     60    - AAC ACA AAA AGA ACT AAA AAA AGC CAA CAT AC - #T AGC GAA GGA ACT TAT     240    Asn Thr Lys Arg Thr Lys Lys Ser Gln His Th - #r Ser Glu Gly Thr Tyr    # 80    - ATC CAT TTC CAA ATA AGT GGC GTT ACA AAT AC - #T GAA AAA TTA CCT ACT     288    Ile His Phe Gln Ile Ser Gly Val Thr Asn Th - #r Glu Lys Leu Pro Thr    #                 95    - CCA ATA GAA CTA CCT TTA AAA GTT AAG GTT CA - #T GGT AAA GAT AGC CCC     336    Pro Ile Glu Leu Pro Leu Lys Val Lys Val Hi - #s Gly Lys Asp Ser Pro    #           110    - TTA AAG TAT TGG CCA AAG TTC GAT AAA AAA CA - #A TTA GCT ATA TCA ACT     384    Leu Lys Tyr Trp Pro Lys Phe Asp Lys Lys Gl - #n Leu Ala Ile Ser Thr    #       125    - TTA GAC TTT GAA ATT CGT CAT CAG CTA ACT CA - #A ATA CAT GGA TTA TAT     432    Leu Asp Phe Glu Ile Arg His Gln Leu Thr Gl - #n Ile His Gly Leu Tyr    #   140    - CGT TCA AGC GAT AAA ACG GGT GGT TAT TGG AA - #A ATA ACA ATG AAT GAC     480    Arg Ser Ser Asp Lys Thr Gly Gly Tyr Trp Ly - #s Ile Thr Met Asn Asp    145                 1 - #50                 1 - #55                 1 -    #60    - GGA TCC ACA TAT CAA AGT GAT TTA TCT AAA AA - #G TTT GAA TAC AAT ACT     528    Gly Ser Thr Tyr Gln Ser Asp Leu Ser Lys Ly - #s Phe Glu Tyr Asn Thr    #               175    - GAA AAA CCA CCT ATA AAT ATT GAT GAA ATA AA - #A ACT ATA GAA GCA GAA     576    Glu Lys Pro Pro Ile Asn Ile Asp Glu Ile Ly - #s Thr Ile Glu Ala Glu    #           190    #          582    Ile Asn    - (2) INFORMATION FOR SEQ ID NO:6:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 194 amino              (B) TYPE: amino acid              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: protein    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:    - Met Thr Asn Asp Asn Ile Lys Asp Leu Leu As - #p Trp Tyr Ser Ser Gly    #                 15    - Ser Asp Thr Phe Thr Asn Ser Glu Val Leu As - #p Asn Ser Leu Gly Ser    #             30    - Met Arg Ile Lys Asn Thr Asp Gly Ser Ile Se - #r Leu Ile Ile Phe Pro    #         45    - Ser Pro Tyr Tyr Ser Pro Ala Phe Thr Lys Gl - #y Glu Lys Val Asp Leu    #     60    - Asn Thr Lys Arg Thr Lys Lys Ser Gln His Th - #r Ser Glu Gly Thr Tyr    # 80    - Ile His Phe Gln Ile Ser Gly Val Thr Asn Th - #r Glu Lys Leu Pro Thr    #                 95    - Pro Ile Glu Leu Pro Leu Lys Val Lys Val Hi - #s Gly Lys Asp Ser Pro    #           110    - Leu Lys Tyr Trp Pro Lys Phe Asp Lys Lys Gl - #n Leu Ala Ile Ser Thr    #       125    - Leu Asp Phe Glu Ile Arg His Gln Leu Thr Gl - #n Ile His Gly Leu Tyr    #   140    - Arg Ser Ser Asp Lys Thr Gly Gly Tyr Trp Ly - #s Ile Thr Met Asn Asp    145                 1 - #50                 1 - #55                 1 -    #60    - Gly Ser Thr Tyr Gln Ser Asp Leu Ser Lys Ly - #s Phe Glu Tyr Asn Thr    #               175    - Glu Lys Pro Pro Ile Asn Ile Asp Glu Ile Ly - #s Thr Ile Glu Ala Glu    #           190    - Ile Asn    - (2) INFORMATION FOR SEQ ID NO:7:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 25 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: DNA (genomic)    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:    #               25 GTCA ACCAG    - (2) INFORMATION FOR SEQ ID NO:8:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 23 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: DNA (genomic)    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:    #                23TCTT TGT    - (2) INFORMATION FOR SEQ ID NO:9:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 22 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: DNA (genomic)    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:    #                 22AAG CG    - (2) INFORMATION FOR SEQ ID NO:10:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 25 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: DNA (genomic)    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:    #               25 TATA AATAG    - (2) INFORMATION FOR SEQ ID NO:11:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 51 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: DNA (genomic)    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:    #             51AGGAGGA AAAAAAAATG TCTACAAACG ATAATATAAA G    - (2) INFORMATION FOR SEQ ID NO:12:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 42 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: DNA (genomic)    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:    #  42              TTAA TTTCTGCTTC TATAGTTTTT AT    - (2) INFORMATION FOR SEQ ID NO:13:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 279 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: DNA (genomic)    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:    - ACCATGAAGA TCTCTGCAGC TGCCCTCACC ATCATCCTCA CTGCAGCCGC CC - #TCTGGGCG      60    - CCCGCGCCTG CCTCACCATA TGGCTCGGAC ACCACTCCCT GCTGCTTTGC CT - #ACCTCTCC     120    - CTCGCGCTGC CTCGTGCCCA CGTCAAGGAG TATTTCTACA CCAGCAGCAA GT - #GCTCCAAT     180    - CTTGCAGTCG TGTTTGTCAC TCGAAGGAAC CGCCAAGTGT GTGCCAACCC AG - #AGAAGAAG     240    #   279            TCAA CTATTTGGAG ATGAGCTAG    __________________________________________________________________________

While various embodiments of the present invention have been describedin detail, it is apparent that modifications and adaptations of thoseembodiments will occur to those skilled in the art. It is to beexpressly understood, however, that such modifications and adaptationsare within the scope of the present invention, as set forth in thefollowing claims:

What is claimed:
 1. A composition, comprising a recombinant constructcomprising a first isolated nucleic acid sequence encoding asuperantigen and a second isolated nucleic acid sequence encoding achemokine, wherein said isolated nucleic acid sequences are operativelylinked to one or more transcription control sequences.
 2. A compositioncomprising:(a) a first recombinant construct comprising an isolatednucleic acid sequence encoding a superantigen operatively linked to oneor more transcription control sequences; and, (b) a second recombinantconstruct comprising an isolated nucleic acid sequence encoding achemokine operatively linked to one or more transcription controlsequences.
 3. A method to treat a mammal that has cancer, said methodcomprising administering to said mammal a therapeutic compositioncomprising:(a) a liposome delivery vehicle; and, (b) a recombinantconstruct comprising a first isolated nucleic acid sequence encoding asuperantigen and a second isolated nucleic acid sequence encoding achemokine, said first and second nucleic acid sequences beingoperatively linked to one or more transcription controlsequences;wherein said first and said second nucleic acid sequencesencoding said superantigen and said chemokine, respectively, arecoexpressed at or adjacent to said cancer; and, wherein saidcoexpression of said superantigen and said chemokine produces a resultselected from the group consisting of alleviation of said cancer,reduction of a tumor associated with said cancer, elimination of a tumorassociated with said cancer, prevention of metastatic cancer, andstimulation of effector cell immunity against said cancer.
 4. A methodto treat a mammal that has cancer, said method comprising administeringto said mammal a therapeutic composition comprising:(a) a liposomedelivery vehicle; (b) a first recombinant construct comprising anisolated nucleic acid sequence encoding a superantigen operativelylinked to one or more transcription control sequences; and, (c) a secondrecombinant construct comprising an isolated nucleic acid sequenceencoding a chemokine operatively linked to one or more transcriptioncontrol sequences;wherein said nucleic acid sequences encoding saidsuperantigen and said chemokine, respectively, are coexpressed at oradjacent to said cancer; and, wherein said coexpression of saidsuperantigen and said chemokine produces a result selected from thegroup consisting of alleviation of said cancer, reduction of a tumorassociated with said cancer, elimination of a tumor associated with saidcancer, prevention of metastatic cancer, and stimulation of effectorcell immunity against said cancer.
 5. A method to treat a mammal thathas cancer, said method comprising;(a) removing cells of said mammal;(b) transfecting said cells in vitro with a recombinant constructcomprising a first isolated nucleic acid sequence encoding asuperantigen and a second isolated nucleic acid sequence encoding achemokine, said first and second nucleic acid sequences beingoperatively linked to one or more transcription control sequences: and,(c) reintroducing said transfected cells to said mammal;wherein saidfirst and said second nucleic acid sequences encoding said superantigenand said chemokine, respectively, are coexpressed at or adjacent to saidcancer; and, wherein said coexpression of said superantigen and saidchemokine produces a result selected from the group consisting ofalleviation of said cancer, reduction of a tumor associated with saidcancer, elimination of a tumor associated with said cancer, preventionof metastatic cancer, and stimulation of effector cell immunity againstsaid cancer.
 6. A method to treat a mammal that has cancer, said methodcomprising;(a) removing cells of said mammal; (b) transfecting saidcells in vitro with a therapeutic composition comprising:(i) a firstrecombinant construct comprising an isolated nucleic acid sequenceencoding a superantigen operatively linked to one or more transcriptioncontrol sequences; and, (ii) a second recombinant construct comprisingan isolated nucleic acid sequence encoding a chemokine operativelylinked to one or more transcription control sequences; and, (c)reintroducing said transfected cells to said mammal;wherein said nucleicacid sequences encoding said superantigen and said chemokine,respectively, are coexpressed at or adjacent to said cancer; and,wherein said coexpression of said superantigen and said chemokineproduces a result selected from the group consisting of alleviation ofsaid cancer, reduction of a tumor associated with said cancer,elimination of a tumor associated with said cancer, prevention ofmetastatic cancer, and stimulation of effector cell immunity againstsaid cancer.
 7. The composition as in one of claims 1 or 2, wherein saidsuperantigen is selected from the group consisting of staphylococcalenterotoxins, retroviral antigens, streptococcal antigens, mycoplasmaantigens, mycobacteria antigens, viral antigens and protozoan antigens.8. The composition as in one of claims 1 or 2, wherein said superantigencomprises staphylococcal enterotoxins.
 9. The composition as in one ofclaims 1 or 2, wherein said superantigen is selected from the groupconsisting of SEA, SEB, SEC₁, SEC₂, SEC₃, SED, SEE and TSST.
 10. Thecomposition as in one of claims 1 or 2, wherein said superantigen isfrom a virus selected from the group consisting of mouse mammary tumorvirus, rabies virus and herpes virus.
 11. The composition as in one ofclaims 1 or 2, wherein said transcription control sequences are selectedfrom the group consisting of RSV control sequences, CMV controlsequences, retroviral LTR sequences, SV-40 control sequences and β-actincontrol sequences.
 12. The composition as in one of claims 1 or 2,wherein said therapeutic composition further comprises apharmaceutically acceptable carrier selected from the group consistingof an aqueous physiologically balanced solution, an artificiallipid-containing substrate, a natural lipid-containing substrate, anoil, an ester, a glycol, a virus and a metal particle.
 13. Thecomposition of claim 12, wherein said pharmaceutically acceptablecarrier is selected from the group consisting of liposomes, micelles,cells, and an aqueous physiologically balanced solution.
 14. Thecomposition of claim 12, wherein said pharmaceutically acceptablecarrier is a liposome.
 15. The composition as in one of claims 1 or 2,wherein said recombinant construct is dicistronic and comprises an IRES.16. The composition of claim 2, wherein said recombinant constructcomprising a nucleic acid sequence encoding a superantigen is selectedfrom the group consisting of PCR₃ -SEB, PCR₃ -SEA, PCR₃ -SEB.S, PCR₃-SEA.S and PCR₃ -TSST.
 17. The composition of claim 2, wherein saidsecond recombinant construct is selected from the group consisting ofPCR₃ -RANTES, PCR₃ -MIP1α and PCR₃ -MIP1β.
 18. The composition of claim1, wherein said first nucleic acid sequence and said second nucleic acidsequence are separated by an IRES.
 19. The method as in one of claims3-6, wherein said superantigen is selected from the group consisting ofstaphylococcal enterotoxins, retroviral antigens, streptococcalantigens, mycoplasma antigens, mycobacteria antigens, viral antigens andprotozoan antigens.
 20. The method as in one of claims 3-6, wherein saidtranscription control sequences are selected from the group consistingof RSV control sequences, CMV control sequences, retroviral LTRsequences, SV-40 control sequences and β-actin control sequences. 21.The method as in one of claims 3-6, wherein said mammal is a human. 22.The method as in one of claims 3-6, wherein said mammal is selected fromthe group consisting of humans, dogs, cats, sheep, cattle, horses andpigs.
 23. The method as in one of claims 3-6, wherein said cancer isselected from the group consisting of melanomas, squamous cellcarcinoma, breast cancers, head and neck carcinomas, thyroid carcinomas,soft tissue sarcomas, bone sarcomas, testicular cancers, prostaticcancers, ovarian cancers, bladder cancers, skin cancers, brain cancers,angiosarcomas, hemangiosarcomas, mast cell tumors, primary hepaticcancers, lung cancers, pancreatic cancers, gastrointestinal cancers,renal cell carcinomas, and hematopoietic neoplasias.
 24. The method asin one of claims 3-6, wherein said cancer is selected from the groupconsisting of melanomas, lung cancers, thyroid carcinomas, breastcancers, renal cell carcinomas, squamous cell carcinomas, brain tumorsand skin cancers.
 25. The method as in one of claims 3 or 4, whereinsaid liposome delivery vehicle includes a compound which specificallydelivers said liposome to said cancer.
 26. The method as in one ofclaims 3 or 4, wherein said therapeutic composition is administered tosaid mammal at or adjacent to said cancer.
 27. The method as in one ofclaims 4 or 6, wherein said recombinant construct comprising a nucleicacid sequence encoding a superantigen is selected from the groupconsisting of PCR₃ -SEB, PCR₃ -SEA, PCR₃ -SEB.S, PCR₃ -SEA.S and PCR₃-TSST.
 28. The method as in one of claims 4 or 6, wherein said secondrecombinant construct is selected from the group consisting of PCR₃-RANTES, PCR₃ -MIP1α and PCR₃ -MIP1β.